United States Office of Water EPA 823-B-00-007

Environmental Protection (4305) November 2000

Agency

Guidance for Assessing

Chemical Contaminant

Data for Use in Fish

Advisories

Volume 1

Fish Sampling and Analysis

Third Edition
Guidance for Assessing Chemical Contaminant

Data for Use in Fish Advisories

Volume 1: Fish Sampling and Analysis

Third Edition

Office of Science and Technology

Office of Water

U.S. Environmental Protection Agency

Washington, DC
United States

Environmental Protection Agency

(4305)

Washington, DC 20460

Official Business

Penalty for Private Use $300
Guidance for Assessing Chemical Contamin

Volume 1: Fish Sampling and Analysis
TABLE OF CONTENTS

iii

TABLE OF CONTENTS

Section Page

Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Tables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xii

Acronyms ................................................ xiii

Executive Summary ....................................... ES-1

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1

1.1 Historical Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-1

1.1.1 Establishment of the Fish Contaminant Workgroup . . . 1-3

1.1.2 Development of a National Fish Advisory Database . . . 1-3

1.2 Purpose ......................................... 1-10

1.3 Objectives ....................................... 1-13

1.4 Relationship of Manual to Other Guidance Documents ..... 1-15

1.5 Contents of Volume 1 .............................. 1-15

1.6 New Information And Revisions to Volume 1 ............. 1-18

2 Monitoring Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-1

2.1 Screening Studies (Tier 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-4

2.2 Intensive Studies (Tier 2) ............................ 2-13

3 Target Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-1

3.1 Purpose of Using Target Species . . . . . . . . . . . . . . . . . . . . . . 3-1

3.2 Criteria for Selecting Target Species . . . . . . . . . . . . . . . . . . . . 3-2

3.3 Freshwater Target Species . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-3

3.3.1 Target Finfish Species . . . . . . . . . . . . . . . . . . . . . . . . . 3-5

3.3.2 Target Turtle Species ......................... 3-10

3.4 Estuarine/marine Target Species ...................... 3-15

3.4.1 Target Shellfish Species ....................... 3-23

3.4.2 Target Finfish Species ........................ 3-28

4 Target Analytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-1

4.1 Recommended Target Analytes . . . . . . . . . . . . . . . . . . . . . . . 4-1

4.2 Selection and Prioritization of Target Analytes . . . . . . . . . . . . . 4-3

4.3 Target Analyte Profiles ............................. 4-12

4.3.1 Metals .................................... 4-12

4.3.2 Organochlorine Pesticides ..................... 4-24

4.3.3 Organophosphate Pesticides ................... 4-38
Section Page

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iv

4.3.4 Chlorophenoxy Herbicides ..................... 4-43

4.3.5 Polycyclic Aromatic Hydrocarbons ............... 4-44

4.3.6 Polychlorinated Biphenyls (Total) ................ 4-47

4.3.7 Dioxins and Dibenzofurans ..................... 4-54

4.4 Target Analytes Under Evaluation ..................... 4-58

4.4.1 Lead ...................................... 4-58

5 Screening Values for Target Analytes . . . . . . . . . . . . . . . . . . . . . . . 5-1

5.1 General Equations for Calculating Screening Values . . . . . . . . 5-1

5.1.1 Noncarcinogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-3

5.1.2 Carcinogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-3

5.1.3 Recommended Values for Variables in Screening 

Value Equations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-4

5.2 Screening Values for Target Analytes . . . . . . . . . . . . . . . . . . . 5-9

5.3 Comparison of Target Analyte Concentrations with 

Screening Values ................................. 5-16

5.3.1 Metals .................................... 5-16

5.3.2 Organics ................................... 5-18

6 Field Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1

6.1 Sampling Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6-1

6.1.1 Screening Studies (Tier 1) . . . . . . . . . . . . . . . . . . . . . . 6-2

6.1.2 Intensive Studies (Tier 2) ...................... 6-22

6.2 Sample Collection ................................. 6-38

6.2.1 Sampling Equipment and Use .................. 6-38

6.2.2 Preservation of Sample Integrity ................ 6-45

6.2.3 Field Recordkeeping ......................... 6-46

6.3 Sample Handling .................................. 6-55

6.3.1 Sample Selection ............................ 6-55

6.3.2 Sample Packaging ........................... 6-61

6.3.3 Sample Preservation ......................... 6-64

6.3.4 Sample Shipping ............................ 6-65

7 Laboratory Procedures I—Sample Handling . . . . . . . . . . . . . . . . . . 7-1

7.1 Sample Receipt And Chain-of-custody . . . . . . . . . . . . . . . . . . 7-1

7.2 Sample Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7-3

7.2.1 General Considerations . . . . . . . . . . . . . . . . . . . . . . . . 7-3

7.2.2 Processing Fish Samples . . . . . . . . . . . . . . . . . . . . . . . 7-7

7.2.3 Processing Turtle Samples ..................... 7-16

7.2.4 Processing Shellfish Samples .................. 7-24

7.3 Sample Distribution ................................ 7-29

7.3.1 Preparing Sample Aliquots ..................... 7-29

7.3.2 Sample Transfer ............................. 7-32
Section Page

TABLE OF CONTENTS

v

8 Laboratory Procedures II—Sample Analyses . . . . . . . . . . . . . . . . . 8-1

8.1 Recommended Analytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-1

8.1.1 Target Analytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-1

8.1.2 Lipid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-1

8.2 Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-3

8.2.1 Lipid Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-3

8.2.2 Target Analyte Methods . . . . . . . . . . . . . . . . . . . . . . . . 8-4

8.3 Quality Assurance and Quality Control Considerations ..... 8-12

8.3.1 QA Plans .................................. 8-14

8.3.2 Method Documentation ....................... 8-14

8.3.3 Minimum QA and QC Requirements for Sample 

Analyses ................................... 8-14

8.4 Documentation and Reporting of Data .................. 8-46

8.4.1 Analytical Data Reports ....................... 8-46

8.4.2 Summary Reports ........................... 8-47

9 Data Analysis and Reporting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-1

9.1 Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-1

9.1.1 Screening Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-1

9.1.2 Intensive Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-2

9.2 Data Reporting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-3

9.2.1 State Data Reports . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-3

9.2.2 Reports to the National Fish Tissue Residue 

Data Repository (NFTRDR) . . . . . . . . . . . . . . . . . . . . . 9-3

10 Literature Cited ....................................... 10-1

Appendix

A 1993 Fish Contaminant Workgroup . . . . . . . . . . . . . . . . . . . . . . . . . A-1

B Screening Values for Defining Green Areas . . . . . . . . . . . . . . . . . . B-1

C Use of Individual Samples in Fish Contaminant Monitoring

Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1

D Fish and Shellfish Species for which State Consumption

Advisories Have Been Issued . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-1

E Target Analytes Analyzed in National or Regional Monitoring

Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-1

F Pesticides and Herbicides Recommended as Target

Analytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-1
Section Page

TABLE OF CONTENTS

vi

G Target Analyte Dose-Response Variables and Associated

Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G-1

H A Recommended Method for Inorganic Arsenic Analysis . . . . . . . . . H-1

I Quality Assurance and Quality Control Guidance . . . . . . . . . . . . . . I-1

J Recommended Procedures for Preparing Whole Fish Composite

Homogenate Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J-1

K General Procedures for Removing Edible Tissues from

Freshwater Turtles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K-1

L General Procedures for Removing Edible Tissues from Shellfish . . L-1

M Sources of Reference Materials and Standards . . . . . . . . . . . . . . . M-1

N Statistical Methods for Comparing Samples: Spatial and

Temporal Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-1
FIGURES

vii

FIGURES

Number Page

1-1 Total number of fish advisories in effect in each state in 1998 

(change from 1997) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1-6

1-2 Trends in number of advisories issued for various pollutants . . . . . 1-7

1-3 Series summary: Guidance for assessing chemical 

contamination data for use in fish advisories ................ 1-16

2-1 Recommended strategy for State fish and shellfish contaminant

monitoring programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2-3

3-1 Geographic range of the common snapping turtle (Chelydra

serpentina) .......................................... 3-12

3-2 Geographic distributions of three bivalve species used 

extensively in national contaminant monitoring programs ...... 3-27

4-1 States issuing fish and shellfish advisories for mercury ........ 4-21

4-2 States issuing fish and shellfish advisories for chlordane ....... 4-27

4-3 States issuing fish and shellfish advisories for PCBs .......... 4-51

4-4 States issuing fish and shellfish advisories for dioxin/furans .... 4-57

5-1 Recommended Values for Mean Body Weights (BWs)

and Fish Consumption Rates (CRs) for Selected 

Subpopulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-5

5-2 Fish Consumption Rates for Various Fisher Populations ....... 5-10

5-3 Dose-Response Variables and Recommended Screening 

Values (SVs) for Target Analytes - Recreational Fishers ....... 5-11

5-4 Dose-Response Variables and Recommended Screening 

Values (SVs) for Target Analytes - Subsistence Fishers ....... 5-13

5-5 Example Screening Values (SVs) for Various Target

Populations and Risk Levels (RLs) ........................ 5-17

5-6 Toxicity Equivalency Factors for Various PAHs .............. 5-20

5-7 Toxicity Equivalency Factors (TEFs) for Tetra-through OctaChlorinated Dibenzo-p-Dioxins and Dibenzofurans and 

Dioxin-Like PCBs ..................................... 5-22
Number Page

FIGURES

viii

6-1 Example of a sample request form . . . . . . . . . . . . . . . . . . . . . . . . . 6-3

6-2 U.S. fish and wildlife service regions . . . . . . . . . . . . . . . . . . . . . . . 6-7

6-3 Example of a field record for fish contaminant monitoring

program—screening study .............................. 6-47

6-4 Example of a field record for shellfish contaminant monitoring

program—screening study .............................. 6-48

6-5 Example of a field record for fish contaminant monitoring

program—intensive study .............................. 6-49

6-6 Example of a field record for shellfish contaminant monitoring

program—intensive study .............................. 6-51

6-7 Example of a sample identification label .................... 6-52

6-8 Example of a chain-of-custody tag or label ................. 6-53

6-9 Example of a chain-of-custody record form ................. 6-54

6-10 Recommended measurements of body length and size for fish,

shellfish, and turtles ................................... 6-58

7-1 Preparation of fish fillet composite homogenate samples . . . . . . . . 7-8

7-2 Sample processing record for fish contaminant monitoring

program—fish fillet composites. .......................... 7-10

7-3 Illustration of basic fish filleting procedure. .................. 7-13

7-4 Preparation of individual turtle homogenate samples .......... 7-17

7-5 Sample processing record for a contaminant monitoring

program—individual turtle samples ....................... 7-19

7-6 Illustration of basic turtle resection procedure ............... 7-21

7-7 Preparation of shellfish edible tissue composite homogenate 

samples ............................................ 7-25

7-8 Sample processing record for shellfish contaminant monitoring

program—edible tissue composites. ...................... 7-27

7-9 Example of a fish and shellfish monitoring program sample

aliquot record. ....................................... 7-31

7-10 Example of a fish and shellfish monitoring program sample

transfer record ....................................... 7-33

8-1 Recommended contents of analytical standard operating 

procedures (SOPs). ................................... 8-15

9-1 Recommended data reporting requirements for screening and

intensive studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-4

9-2 Key information fields for the National Fish Tissue Residue 

Data Repository . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9-6
TABLES

ix

TABLES

Number Page

1-1 U.S. Advisories Issued from 1993 to 1998 by Type . . . . . . . . . . . . 1-5

1-2 Summary of Statewide Advisories in Effect in 1998 . . . . . . . . . . . . 1-9

1-3 Comparison of FDA Action Levels and Tolerances with EPA 

Screening Values ..................................... 1-12

2-1 Recommended Strategy for State Fish and Shellfish

Contaminant Monitoring Programs . . . . . . . . . . . . . . . . . . . . . . . . . 2-5

3-1 Recommended Target Species for Inland Fresh Waters . . . . . . . . 3-4

3-2 Recommended Target Species for Great Lakes Waters . . . . . . . . 3-4

3-3 Comparison of Freshwater Finfish Species Used in Several 

National Fish Contaminant Monitoring Programs . . . . . . . . . . . . . . 3-6

3-4 Freshwater Turtles Recommended for Use as Target Species . . . 3-7

3-5 Average Fish Tissue Concentrations (ppb) of Xenobiotics for 

Major Finfish Species Sampled in the National Study of 

Chemical Residues in Fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3-8

3-6 Average Fish Tissue Concentrations (ppt) of Dioxins and 

Furans for Major Finfish Species Sampled in the National 

Study of Chemical Residues in Fish . . . . . . . . . . . . . . . . . . . . . . . . 3-9

3-7 Principal Freshwater Fish Species Cited in State Fish

Consumption Advisories ................................ 3-11

3-8 Principal Freshwater Turtle Species Cited in State 

Consumption Advisories ................................ 3-12

3-9 Studies Using Freshwater Turtles as Biomonitors of 

Environmental Contamination ........................... 3-14

3-10 Recommended Target Species for Northeast Atlantic

Estuaries and Marine Waters (Maine through Connecticut) ..... 3-16

3-11 Recommended Target Species for Mid-Atlantic Estuaries 

and Marine Waters (New York through Virginia) ............. 3-17

3-12 Recommended Target Species for Southeast Atlantic

Estuaries and Marine Waters (North Carolina through Florida) . . 3-18

3-13 Recommended Target Species for Gulf of Mexico Estuaries 

and Marine Waters (West Coast of Florida through Texas) ..... 3-19

3-14 Recommended Target Species for Pacific Northwest 

Estuaries and Marine Waters (Alaska through Oregon) ........ 3-20

3-15 Recommended Target Species for Northern California 

Estuaries and Marine Waters (Klamath River through 

Morro Bay) .......................................... 3-21
Number Page

TABLES

x

3-16 Recommended Target Species for Southern California

Estuaries and Marine Waters (Santa Monica Bay to 

Tijuana Estuary) ...................................... 3-22

3-17 Sources of Information on Commercial and Sportfishing

Species in Various Coastal Areas of the United States ........ 3-24

3-18 Estuarine/Marine Species Used in Several National Fish 

and Shellfish Contaminant Monitoring Programs ............. 3-25

3-19 Principal Estuarine/Marine Fish and Shellfish Species Cited 

in State Consumption Advisories ......................... 3-29

4-1 Recommended Target Analytes . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-2

4-2 Contaminants Resulting in Fish and Shellfish Advisories . . . . . . . . 4-4

4-3 Selection and Prioritization of Target Analytes by Watershed 

Type . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4-6

4-4 Total Mercury and Methylmercury Concentrations in Estuarine 

Fish from South Florida ................................ 4-18

4-5 Chlordane Constituent Concentrationsa

 Detected in the EPA 

National Study of Chemical Residues in Fish ................ 4-26

4-6 Summary of PCBs Detected in Fish Tissuea

 as Part of the 

National Study of Chemical Residues in Fish ................ 4-50

4-7 Polychlorinated Biphenyl (PCB) Congeners Recommended for

Quantitation as Potential Target Analytes .................. 4-53

4-8 Summary of Dioxins/Furans Detected in Fish Tissue as Part 

of the EPA National Study of Chemical Residues in Fish ....... 4-56

4-9 Dibenzo-p-Dioxins and Dibenzofurans Recommended for 

Analysis as Target Analytes ............................. 4-59

5-1 Recommended Values for Mean Body Weights (BWs)

and Fish Consumption Rates (CRs) for Selected 

Subpopulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5-5

5-2 Fish Consumption Rates for Various Fisher Populations ....... 5-10

5-3 Dose-Response Variables and Recommended Screening 

Values (SVs) for Target Analytes - Recreational Fishers ....... 5-11

5-4 Dose-Response Variables and Recommended Screening 

Values (SVs) for Target Analytes - Subsistence Fishers ....... 5-13

5-5 Example Screening Values (SVs) for Various Target 

Populations and Risk Levels (RLs) ........................ 5-17

5-6 Toxicity Equivalency Factors for Various PAHs 5-20

5-7 Toxicity Equivalency Factors (TEFs) for Tetra-through 

Octa-Chlorinated Dibenzo-p-Dioxins and Dibenzofurans

and Dioxin-Like PCBs ................................. 5-22
Number Page

TABLES

xi

6-1 Values of for Various Combinations of n and m . . 6-30 2

n m (n 1). 2 2

1/2

-

é

ë

ê ù

û

ú

6-2 Estimates of Statistical Power of Hypothesis of Interest Under

Specified Assumptions ................................. 6-32

6-3 Observed Ratios (/SV) of Selected Target Analytes .......... 6-33

6-4 Summary of Fish Sampling Equipment .................... 6-39

6-5 Summary of Shellfish Sampling Equipment ................. 6-40

6-6 Checklist of Field Sampling Equipment and Supplies for 

Fish and Shellfish Contaminant Monitoring Programs ......... 6-42

6-7 Safety Considerations for Field Sampling Using a Boat ........ 6-43

6-8 Recommendations for Preservation of Fish, Shellfish, and Turtles

Samples from Time of Collection to Delivery at the Processing

Laboratory .......................................... 6-63

7-1 Recommendations for Container Materials, Preservation, and

Holding Times for Fish, Shellfish, and Turtle Tissues from

Receipt at Sample Processing Laboratory to Analysis . . . . . . . . . . 7-4

7-2 Weights (g) of Individual Homogenates Required for

Screening Study Composite Homogenate Sample ............ 7-16

7-3 Recommended Sample Aliquot Weights and Containers for

Various Analyses ..................................... 7-30

8-1 Contract Laboratories Conducting Dioxin/Furan Analyses in

Fish and Shellfish Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-2

8-2 Current References for Analytical Methods for Contaminants

in Fish and Shellfish Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8-5

8-3 Recommended Analytical Techniques for Target Analytes . . . . . . 8-8

8-4 Range of Detection and Quantitation Limits of Current

Analytical Methods for Recommended Target Analytes ........ 8-10

8-5 Approximate Range of Costs per Sample for Analysis of

Recommended Target Analytes .......................... 8-13

8-6 Recommended Quality Assurance and Quality Control

Samples ............................................ 8-17

8-7 Minimum Recommended QA and QC Samples for Routine

Analysis of Target Analytes ............................. 8-23

8-8 Fish and Shellfish Tissue Reference Materials ............... 8-26
ACKNOWLEDGMENTS

xii

ACKNOWLEDGMENTS

This report was prepared by the U.S. Environmental Protection Agency, Office of

Water, Fish and Wildlife Contamination Program. The EPA Project Manager for

this document was Jeffrey Bigler who provided overall project coordination as well

as technical direction. EPA was supported in the development of this document

by the Research Triangle Institute (RTI) (EPA Contract Number 68-C7-0056). Pat

Cunningham of RTI was the contractor’s Project Manager. Preparation of the

First Edition of this guidance in 1993 was facilitated by the substantial efforts of

the numerous Workgroup members and reviewers listed in Appendix A. 
ACRONYMS

xiii

ACRONYMS

AFS American Fisheries Society

ANOVA Analysis of Variance 

ATSDR Agency for Toxic Substances and Disease Registry 

BCF bioconcentration factor 

BW body weight 

CDD chlorodibenzo-p-dioxin

CDF chlorodibenzofuran

CERCLA Comprehensive Environmental Response, Compensation,

and Liability Act 

CLP Contract Laboratory Program

COC chain-of-custody 

CR consumption rate 

CRM certified reference material

CRADAs Cooperative Research and Development Agreements 

CSF cancer slope factor

CSFII Continuing Survey of Food Intake by Indivdiuals

CSOs combined sewer overflows 

CV coefficient of variation

CVAAS cold vapor atomic absorption spectrometry

DFTPP decafluorotriphenylphosphine

DOT U.S. Department of Transportation 

EMAP Environmental Monitoring and Assessment Program

EMAP-NC Environmental Monitoring and Assessment Program—Near 

Coastal

EPA U.S. Environmental Protection Agency 

EMMI Environmental Monitoring Methods Index System

FDA U.S. Food and Drug Administration 
ACRONYMS

xiv

FWS U.S. Fish and Wildlife Service 

-BHC benzene hexachloride 

-HCH hexachlorocyclohexane 

GC/ECD gas chromatography/electron capture detection 

GC/FID gas chromatography/flame ionization detection

GC/FPD gas chromatography/flame photometric detection

GC/NPD gas chromatography/nitrogen-phosphorus detection

GC/MS gas chromatography/mass spectrometry 

GFAA graphic furnace atomic absorption spectrometry

GLIFWC Great Lakes Indian Fish and Wildlife Commission

GPS Global Positioning System 

HAA hydride generation atomic absorption spectrometry

HEAST Health Effects Assessment Summary Tables

HPLC/MS high-performance liquid chromatography/mass spectrometry

HRGC/LRMS high-resolution gas chromography/low-resolution mass

spectrometry

HRGC/HRMS high-resolution gas chromatography/high-resolution mass

spectrometry

ICP inductively coupled plasma atomic emission spectrometry

IDL instrument detection limit

IRIS Integrated Risk Information System 

IUPAC Information Union of Pure and Applied Chemistry

LAN local area network

LLD lower limits of detection

LOAEL lowest observed adverse effects level

LOD limit of detection

LOQ limit of quantitation

MDL method detection limit 

MQL method quantitation limit 

NAS National Academy of Sciences

NEP National Estuary Program

NERRS National Estuarine Research Research System
ACRONYMS

xv

NCBP National Contaminant Biomonitoring Program 

NCR no-carbon-required 

ND not detected

NEP National Estuary Program

NFTDR National Fish Tissue Data Repository 

NIST National Institute of Standards and Technology 

NLFWA National Listing of Residue Fish and Wildlife Advisors

NOAA National Oceanic and Atmospheric Administration 

NOAEL no observed adverse effects level

NRCC National Research Council of Canada

NS&T National States and Trends Program

NSCRF National Study of Chemical Residues in Fish

NTIS National Technical Information Service

OAPCA Organotin Antifouling Paint Control Act 

OAQPS Office of Air Quality Planning and Standards 

OCDD octachlorodibenzo-p-dioxin

OCDF octachlorodibenzofuran

ODES Ocean Discharge Evaluation System 

ODW Office of Drinking Water 

OHEA Office of Health and Environmental Assessment 

OPP Office of Pesticide Programs 

ORSANCO Ohio River Valley Water Sanitation Commission 

PAB Population Adjusted Dose

PAHs polycyclic aromatic hydrocarbons 

PBBs polybrominated biphenyls

PCBs polychlorinated biphenyls 

PCDDs polychlorinated dibenzo-p-dioxins 

PCDFs polychlorinated dibenzofurans 

PEC potency equivalency concentration 

PNAs polynuclear aromatic hydrocarbons 

PQL practical quantitation limit

PTFE polytetrafluoroethylene 
ACRONYMS

xvi

QA quality assurance 

QC quality control 

RCRA Resource Conservation and Recovery Act 

RDL reliable detection limit

RFs response factors

RfD reference dose 

RL risk level

RPs relative potencies 

RPD relative percent difference

RRFs relative response factors

RSD relative standard deviation

SOPs standard operating procedures 

SRMs standard reference materials

SVs screening values 

2,4,5-T 2,4,5-trichlorophenoxyacetic acid 

2,3,7,8-TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin 

2,3,7,8-TCDF 2,3,7,8-tetrachlorodibenzofuran 

2,4,5-T 2,4,5-trichlorophenoxyacetic acid

2,4,5-TCP 2,4,5-trichlorophenol 

TEFs toxicity equivalency factor

TEQs toxicity equivalency concentrations 

TVA Tennessee Valley Authority 

UF uncertainty factor

UM modifying factor

USDA U.S. Department of Agriculture 

USGS U.S. Geological Survey 

USFWS U.S. Fish and Wildlife Service

WHO World Health Organization
EXECUTIVE SUMMARY

ES-1

EXECUTIVE SUMMARY

A 1988 survey, funded by the U.S. Environmental Protection Agency (EPA) and

conducted by the American Fisheries Society, identified the need for standardizing

the approaches to evaluating risks and developing fish consumption advisories

that are comparable across different jurisdictions. Four major components were

identified as critical to the development of a consistent risk-based approach:

standardized practices for sampling and analyzing fish, standardized risk

assessment methods, standardized procedures for making risk management

decisions, and standardized approaches for communicating risk to the general

public.

To address concerns raised by the survey respondents, EPA began developing

a series of four documents designed to provide guidance to state, local, regional,

and tribal environmental health officials responsible for designing contaminant

monitoring programs and issuing fish and shellfish consumption advisories. It is

essential that all four documents be used together, since no single volume

addresses all of the topics involved in the development of fish consumption

advisories. The documents are meant to provide guidance only and do not

constitute a regulatory requirement. This document series includes:

Guidance for Assessing Chemical Contaminant Data for Use in Fish Advisories

Volume 1: Fish Sampling and Analysis

Volume 2: Risk Assessment and Fish Consumption Limits

Volume 3: Overview of Risk Management

Volume 4: Risk Communication.

Volume 1 was first released in September 1993 and was followed by a second

edition in September 1995. This current revision to the Volume 1 guidance

provides the latest information on sampling and analysis procedures based on

new information provided by EPA. The major objective of Volume 1 is to provide

information on sampling strategies for a contaminant monitoring program. In

addition, information is provided on selection of target species; selection of

chemicals as target analytes; development of human health screening values;

sample collection procedures including sample processing, sample preservation,

and shipping; sample analysis; and data reporting and analysis.

Volume 2 was first released in June 1994 and was followed by a second edition

in July 1997. A third edition will be released in November 2000. This volume

provides guidance on the development of appropriate meal sizes and frequency

of meal consumption (e.g., one meal per week) for the target analytes that
EXECUTIVE SUMMARY

ES-2

bioaccumulate in fish tissues. In addition to the presentation of consumption

limits, Volume 2 contains a discussion of risk assessment methods used to derive

the consumption limits as well as a discussion of methods to modify these limits

to reflect local conditions. Volume 2 also contains toxicological profiles for each

of the 25 target analytes.

Volume 3 was published in June 1996 and provides an overview of a risk

management framework. This volume provides information on selecting and

implementing various options for reducing health risks associated with the

consumption of chemically contaminated fish and shellfish. Using a human health

risk-based approach, states can determine the level of the advisory and the most

appropriate type of advisory to issue. Methods to evaluate population risks for

specific groups, waterbodies, and geographic areas are also presented.

Volume 4 was published in March 1995 and provides guidance on risk communication as a process for sharing information with the public on the health risks of

consuming chemically contaminated fish and shellfish. This volume provides

guidance on problem analysis and program objectives, audience identification

and needs assessments, communication strategy design, implementation and

evaluation, and responding to public inquiries. 

EPA welcomes your suggestions and comments. A major goal of this guidance

document series is to provide a clear and usable summary of critical information

necessary to make informed decisions concerning the development of fish

consumption advisories. We encourage comments and hope this document will

be a useful adjunct to the resources used by the states, local governments, and

tribal organizations in making decisions concerning the development of fish

advisories within their various jurisdictions.
1. INTRODUCTION

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SECTION 1

INTRODUCTION

1.1 HISTORICAL PERSPECTIVE

Contamination of aquatic resources, including freshwater, estuarine, and marine

fish and shellfish, has been documented in the scientific literature for many

regions of the United States (NAS, 1991). Environmental concentrations of some

pollutants have decreased over the past 25 years as a result of better water

quality management practices. However, environmental concentrations of other

heavy metals, pesticides, and toxic organic compounds have increased due to

intensifying urbanization, industrial development, and use of new agricultural

chemicals. Our Nation’s waterbodies are among the ultimate repositories of

pollutants released from these activities. Pollutants come from permitted point

source discharges (e.g., industrial and municipal facilities), accidental spill events,

and nonpoint sources (e.g., agricultural practices, resource extraction, urban

runoff, in-place sediment contamination, groundwater recharge, vehicular

exhaust, and atmospheric deposition from various combustion and incineration

processes).

Once these toxic contaminants reach surface waters, they may concentrate

through aquatic food chains and bioaccumulate in fish and shellfish tissues.

Aquatic organisms may bioaccumulate environmental contaminants to more than

1,000,000 times the concentrations detected in the water column (U.S. EPA,

1992c, 1992d). Thus, fish and shellfish tissue monitoring serves as an important

indicator of contaminated sediments and water quality problems, and many states

routinely conduct chemical contaminant analyses of fish and shellfish tissues as

part of their comprehensive water quality monitoring programs (Cunningham and

Whitaker, 1989; Cunningham, 1998; Cunningham and Sullivan,1999). Tissue

contaminant monitoring also enables state agencies to detect levels of contamination in fish and shellfish tissue that may be harmful to human consumers. If states

conclude that consumption of chemically contaminated fish and shellfish poses

an unacceptable human health risk, they may issue local fish consumption

advisories or bans for specific waterbodies and specific fish and shellfish species

for specific populations.

In 1989, the American Fisheries Society (AFS), at the request of the U.S.

Environmental Protection Agency (EPA), conducted a survey of state fish and

shellfish consumption advisory practices. Questionnaires were sent to health

departments, fisheries agencies, and water quality/environmental management

departments in all 50 states and the District of Columbia. Officials in all 50 states

and the District responded.
1. INTRODUCTION

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Respondents were asked to provide information on several issues including

• Agency responsibilities

• Sampling strategies

• Sample collection procedures

• Chemical residue analysis

procedures

• Risk assessment methodologies

• Data interpretation and advisory

development

• State concerns 

• Recommendations for federal

assistance.

Cunningham et al. (1990) summarized the survey responses and reported that

monitoring and risk assessment procedures used by states in their fish and

shellfish advisory programs varied widely. States responded to the question

concerning assistance from the federal government by requesting that federal

agencies

• Provide a consistent approach for state agencies to use in assessing health

risks from consumption of chemically contaminated fish and shellfish

• Develop guidance on sample collection procedures

• Develop and/or endorse uniform, cost-effective analytical methods for

quantitation of contaminants

• Establish a quality assurance (QA) program that includes use of certified

reference materials for chemical analyses.

In March 1991, the National Academy of Sciences (NAS) published a report

entitled Seafood Safety (NAS, 1991) that reviewed the nature and extent of public

health risks associated with seafood consumption and examined the scope and

adequacy of current seafood safety programs. After reviewing over 150 reports

and publications on seafood contamination, the NAS Institute of Medicine

concluded that high concentrations of chemical contaminants exist in various fish

species in a number of locations in the country. The report noted that the fish

monitoring data available in national and regional studies had two major

shortcomings that affected their usefulness in assessing human health risks:

• In some of the more extensive studies, analyses were performed on nonedible

portions of finfish (e.g., liver tissue) or on whole fish, which precludes accurate

determination of human exposures.

• Studies did not use consistent methods of data reporting (e.g., both geometric

and arithmetic means were reported in different studies) or failed to report

crucial information on sample size, percent lipid, mean values of contaminant

concentrations, or fish size, thus precluding direct comparison of the data from

different studies and complicating further statistical analysis and risk

assessment.
1. INTRODUCTION

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1.1.1 Establishment of the Fish Contaminant Workgroup

As a result of NAS concerns and state concerns expressed in the AFS survey,

EPA’s Office of Water established a Fish Contaminant Workgroup. It was

composed of representatives from EPA and the following state and federal

agencies:

• U.S. Food and Drug Administration (FDA)

• U.S. Fish and Wildlife Service (FWS)

• Ohio River Valley Water Sanitation Commission (ORSANCO)

• National Oceanic and Atmospheric Administration (NOAA)

• Tennessee Valley Authority (TVA)

• United States Geological Survey (USGS)

and representatives from 26 states: Alabama, Arkansas, California, Colorado,

Delaware, Florida, Georgia, Illinois, Indiana, Louisiana, Maryland, Massachusetts,

Michigan, Minnesota, Missouri, Nebraska, New Hampshire, New Jersey, New

York, North Carolina, North Dakota, Ohio, Oregon, Texas, Virginia, and

Wisconsin.

The objective of the EPA Fish Contaminant Workgroup was to formulate guidance

for states on how to sample and analyze chemical contaminants in fish and

shellfish where the primary end uses of the data included development of fish

consumption advisories. The Workgroup compiled documents describing

protocols currently used by various federal agencies, EPA Regional offices, and

states that have extensive experience in fish contaminant monitoring. Using

these documents, they selected methods considered most cost-effective and

scientifically sound for sampling and analyzing fish and shellfish tissues. These

methods were recommended as standard procedures for use by the states and

are described in this guidance document.

1.1.2 Development of a National Fish Advisory Database

In addition to initiating work on the national guidance document series in 1993,

EPA also initiated work on the development of a national database — The

National Listing of Fish and Wildlife Advisories (NLFWA) database — for tracking

fish and wildlife advisories issued by the states. The 1998 update of the NLFWA

database includes all available information describing state, territorial, tribal, and

federal fish consumption advisories issued in the United States (U.S. EPA 1999a,

1999c). The database contains fish consumption advisory information provided

to EPA by the states and other jurisdictions from 1993 through December 1998.

It also includes information from 1996 through 1997 for 12 Canadian provinces

and territories. No updates to information on Canadian advisories were made in

1998. Since the release of the first fish advisory results in 1994, advisory results

and trends have been accessible to states, territories, tribal organizations, and the

general public by querying the NLFWA database or through summary information

reported each year in the EPA Fact Sheet—Update: National Listing of Fish and
1. INTRODUCTION

1-4

Wildlife Advisories. Fish advisory results and trends reported in the 1999 Fish

Advisory Fact Sheet (U.S. EPA, 1999c) are presented below. The most recent

updates of the Fish Advisory Fact Sheet are available on the EPA website at

http://epa.gov/OST/fish. 

1.1.2.1 Background—

The states, U.S. territories, and Native American tribes (hereafter referred to as

states) have primary responsibility for protecting residents from the health risks

of consuming contaminated noncommercially caught fish and wildlife. They do this

by issuing consumption advisories for the general population, including recreational and subsistence fishers, as well as for sensitive subpopulations (such as

pregnant women, nursing mothers, and children). These advisories inform the

public that high concentrations of chemical contaminants (e.g., mercury and

dioxins) have been found in local fish and wildlife. The advisories include

recommendations to limit or avoid consumption of certain fish and wildlife species

from specified waterbodies or, in some cases, from specific waterbody types (e.g.,

all inland lakes). Similarly, in Canada, the provinces and territories have primary

responsibility for issuing fish consumption advisories for their residents. 

States typically issue five major types of advisories and bans to protect both the

general population and specific subpopulations. 

• When levels of chemical contamination pose a health risk to the general

public, states may issue a no consumption advisory for the general population.

• When contaminant levels pose a health risk to sensitive subpopulations,

states may issue a no consumption advisory for the sensitive subpopulation.

• In waterbodies where chemical contamination is less severe, states may issue

an advisory recommending that either the general population or a sensitive

subpopulation restrict their consumption of the specific species for which the

advisory is issued. 

• The fifth type of state-issued advisory is the commercial fishing ban, which

prohibits the commercial harvest and sale of fish, shellfish, and/or wildlife

species from a designated waterbody and, by inference, the consumption of

all species identified in the fishing ban from that waterbody. 

As shown in Table 1-1, advisories of all types increased overall in number from

1993 to 1998. 

1.1.2.2 Advisories in Effect—

The database includes information on

• Species and size ranges of fish and/or wildlife sampled

• Chemical contaminants identified in the advisory
1. INTRODUCTION

1-5

• Geographic location of each advisory (including narrative information on

landmarks, river miles, or latitude and longitude coordinates of the affected

waterbody and map showing location of waterbody)

• Lake acreage or river miles under advisory

• Population for whom the advisory was issued

• Fish tissue chemical residue data from waterbodies under advisory.

The 1994, 1995, 1996, 1997, and 1998 versions of the NLFWA database can

generate national, regional, and state maps that illustrate any combination of

these advisory parameters. In addition, the 1996 through 1998 versions of the

database can provide information on the percentage of waterbodies in each state

currently under an advisory and the percentage of waters assessed. A new

feature of the 1998 database provides users access to fish tissue residue data for

those waterbodies under advisory in 16 states. The name of each state contact,

phone number, FAX number, and e-mail address are also provided so that users

can obtain additional information concerning specific advisories. Comparable

advisory information (excluding tissue residue data) and contact information for

1996 and 1997 are provided for each Canadian province or territory.

1.1.2.3 Advisory Trends—

The number of waterbodies in the United States under advisory reported in 1998

(2,506) represents a 9% increase from the number reported in 1997 (2,299

advisories) and a 98% increase from the number of advisories issued since 1993

(1,266 advisories). Figure 1-1 shows the number of advisories in effect for each

state in 1998 and the number of advisories issued or rescinded since 1997. The

increase in advisories issued by the states generally reflects an increase in the

number of assessments of the levels of chemical contaminants in fish and wildlife

tissues. These additional assessments were conducted as a result of the

increased awareness of health risks associated with the consumption of

chemically contaminated fish and wildlife. Some of the increase in advisory

numbers, however, may be due to the increasing use of EPA risk assessment

procedures in setting advisories rather than FDA action levels developed for

commercial fisheries. 

Table 1-1. U.S. Advisories Issued from 1993 to 1998 by Type

1993 1994 1995 1996 1997 1998

No Consumption – General Population 503 462 463 563 545 532

No Consumption – Sensitive

Subpopulation

555 720 778 1,022 1,119 1,211

Restricted Consumption – General

Population

993 1,182 1,372 1,763 1,843 2,062

Restricted Consumption – Sensitive

Subpopulation

689 900 1,042 1,370 1,450 1,595

Commercial Fishing Ban 30 30 55 50 52 50

Source: U.S. EPA 1999a, 1999c.
1. INTRODUCTION

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1.1.2.4 Bioaccumulative Pollutants—

Although U.S. advisories have been issued for a total of 46 chemical contaminants, most advisories issued have involved five primary contaminants. These

chemical contaminants are biologically accumulated in the tissues of aquatic

organisms at concentrations many times higher than concentrations in the water.

In addition, these chemical contaminants persist for relatively long periods in

sediments where they can be accumulated by bottom-dwelling organisms and

passed up the food chain to fish. Concentrations of these contaminants in the

tissues of aquatic organisms may be increased at each successive level of the

food chain. As a result, top predators in a food chain, such as largemouth bass,

salmon, or walleye, may have concentrations of these chemicals in their tissues

that can be a million times higher than the concentrations in the water. Mercury,

PCBs, chlordane, dioxins, and DDT (and its degradation products, DDE and DDD)

were at least partly responsible for 99 percent of all fish consumption advisories

in effect in 1998. (See Figure 1-2.)

Source: U.S. EPA, 1999c.

Figure 1-1. Total number of fish advisories in effect in each state in 1998

(change from 1997).
1. INTRODUCTION

1-7

Source: U.S. EPA, 1999a, 1999c.

Figure 1-2. Trends in number of advisories issued for various pollutants.

1.1.2.5 Wildlife Advisories—

In addition to advisories for fish and shellfish, the database also contains several

wildlife advisories. Four states have issued consumption advisories for turtles:

Arizona (3), Massachusetts (1), Minnesota (8), and New York (statewide

advisory). One state (Massachusetts) has an advisory for frogs, New York has a

statewide advisory for waterfowl (including mergansers), Arkansas has an

advisory for woodducks, and Utah has an advisory for American coot and ducks.

Maine issued a statewide advisory for moose liver and kidneys due to cadmium

levels. No new wildlife advisories were issued in 1998. 

1.1.2.6 1998 United States Advisories—

The 1998 database lists 2,506 advisories in 47 states, the District of Columbia,

and the U.S. Territory of American Samoa. Some of these advisories represent

statewide advisories for certain types of waterbodies (e.g., lakes, rivers, and/or

coastal waters). An advisory may represent one waterbody or one type of waterbody within a state’s jurisdiction. Statewide advisories are counted as one

advisory. The database counts one advisory for each waterbody name or type of

waterbody regardless of the number of fish or wildlife species that are affected or

the number of chemical contaminants detected at concentrations of human health

concern. Eighteen states (Alabama, Connecticut, District of Columbia, Florida,

Indiana, Louisiana, Maine, Massachusetts, Michigan, Mississippi, New

Hampshire, New Jersey, New York, North Carolina, Ohio, Rhode Island, Texas,
1. INTRODUCTION

1-8

and Vermont) currently have statewide advisories in effect (see Table 1-2).

Missouri rescinded its statewide advisories for lakes and rivers in 1998, and

Mississippi added a statewide coastal advisory for mercury. A statewide advisory

is issued to warn the public of the potential for widespread contamination of

certain species of fish in certain types of waterbodies (e.g., lakes, rivers and

streams, or coastal waters) or certain species of wildlife (e.g., moose or

waterfowl). In such a case, the state may have found a level of contamination of

a specific pollutant in a particular fish or wildlife species over a relatively wide

geographic area that warrants advising the public of the situation.

The statewide advisories and 2,506 specifically named waterbodies represent

approximately 15.8 percent of the Nation’s total lake acreage and 6.8% of the

Nation’s total river miles. In addition, 100 percent of the Great Lakes waters and

their connecting waters are also under advisory due to one or more contaminants

(e.g., PCBs, dioxins, mercury, and/or chlordane). The Great Lakes waters are

considered separately from other lakes, and their connecting waters are

considered separately from other river miles.

Several states also have issued fish advisories for all of their coastal waters.

Using coastal mileages calculated by the National Oceanic and Atmospheric

Administration (NOAA), an estimated 58.9 percent of the coastline of the

contiguous 48 states currently is under advisory. This includes 61.5 percent of the

Atlantic Coast and 100 percent of the Gulf Coast. No Pacific Coast state has

issued a statewide advisory for any of its coastal waters although several

localized areas along the Pacific Coast are under advisory. The Atlantic coastal

advisories have been issued for a wide variety of chemical contaminants including

mercury, PCBs, dioxins, and cadmium, while all of the Gulf Coast advisories have

been issued for mercury. 

1.1.2.7 Database Use and Access—

The NLFWA database was developed by EPA to help federal, state, and local

government agencies and Native American tribes assess the potential for human

health risks associated with consumption of chemical contaminants in

noncommercially caught fish and wildlife. The data contained in this database

may also be used by the general public to make informed decisions about the

waterbodies in which they choose to fish or harvest wildlife; the frequency with

which they fish these waterbodies; the species, size, and number of fish they

collect; and the frequency with which they consume fish from specific waterbodies. Note: State fish advisory contact information and hyperlinks to state fish

advisory websites are also provided.

EPA provides this 1998 update of the NLFWA database available on the Internet

at

http://www.epa.gov/OST/fish
1. INTRODUCTION

1-9

Further information on specific advisories within a particular state is available from

the appropriate state agency contact listed in the database. This is particularly

important for advisories recommending that consumers restrict their consumption

of fish from certain waterbodies. State health departments provide more specific

information for restricted consumption advisories (RGP and RSP) on the

appropriate meal size and meal frequency (number of meals per week or month)

that is considered safe to consume for a specific consumer group (e.g., the

general public versus pregnant women, nursing mothers, and young children). For

further information on Canadian advisories, contact the appropriate Province

contact given in the database.

For more information concerning the National Fish and Wildlife Contamination

Program, contact:

U.S. Environmental Protection Agency

Office of Science and Technology

National Fish and Wildlife Contamination Program—4305

1200 Pennsylvania Avenue, NW

Washington, DC 20460

Phone 202 260-7301 FAX 202 260-9830

e-mail: Bigler.Jeff@epa.gov 

Table 1-2. Summary of Statewide Advisories in Effect in 1998

State Lakes Rivers Coastal Waters

Alabama — — Mercury

Connecticut Mercury Mercury PCBs

District of Columbia PCBs PCBs —

Florida — — Mercury

Indiana — Mercury PCBs —

Louisiana — — Mercury

Maine Mercury Mercury Dioxins

Massachusetts Mercury Mercury PCBs

Organics

Michigan Mercury — —

Mississippi — — Mercury

New Hampshire Mercury Mercury PCBs

New Jersey Mercury Mercury PCBs

Cadmium

Dioxins

New York PCBs

Chlordane

Mirex

DDT

PCBs

Chlordane

Mirex

DDT

PCBs

Cadmium

Dioxins

North Carolina Mercury Mercury —

Ohio Mercury Mercury —

Rhode Island — — PCBs

Texas — — Mercury

Vermont Mercury Mercury —

Source: U.S. EPA, 1999a, 1999c.
1. INTRODUCTION

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1.2 PURPOSE

The purpose of this manual is to provide overall guidance to states on methods

for sampling and analyzing contaminants in fish and shellfish tissue that will

promote consistency in the data they use to determine the need for fish consumption advisories. This manual provides guidance only and does not constitute a

regulatory requirement for the states. It is intended to describe what EPA

believes to be scientifically sound methods for sample collection, chemical

analyses, and statistical analyses of fish and shellfish tissue contaminant data for

use in fish contaminant monitoring programs that have as their objective the

protection of public health. This nonregulatory, technical guidance manual is

intended for use as a handbook by state and local agencies that are responsible

for sampling and analyzing fish and shellfish tissue. Adherence to this guidance

will enhance the comparability of fish and shellfish contaminant data, especially

in interstate waters and thus provide more standardized information on fish

contamination problems. 

It should be noted that the EPA methodology described in Volumes 1 and 2 of this

guidance series offers great flexibility to state users. These documents are

designed to meet the objectives of state monitoring and risk assessment

programs by providing options to meet specific state or study needs within state

budgetary constraints. The users of this fish advisory guidance document should

recognize that it is the consistent application of the EPA methodology and

processes rather than individual elements of the program sampling design that

are of major importance in improving consistency among state fish advisory

programs. For example, whether a state elects to collect three composite

samples of five individual fish or four composite samples of eight individual fish

as the basis of its state program is of less importance than a state designing and

executing its monitoring program with attention to all elements of the EPA

methodology having been considered and addressed during the planning and

implementation phases. 

One major factor currently affecting the comparability of fish advisory information

nationwide, is the fact that the states employ different methodologies to determine

the necessity for issuing an advisory. For example, some states currently do not

use the EPA methodology at all or use it only in their assessment of health risks

for certain chemical contaminants. Often these states rely instead on exceedances of FDA action levels or tolerances to determine the need to issue an

advisory. FDA’s mission is to protect the public health with respect to levels of

chemical contaminants in all foods, including fish and shellfish sold in interstate

commerce. FDA has developed both action levels and tolerances to address

levels of contamination in foods. FDA may establish an action level when food

contains a chemical from sources of contamination that cannot be avoided even

by adherence to good agricultural or manufacturing practices, such as 
1. INTRODUCTION

1-11

contamination by a pesticide that persists in the environment. An action level is

an administrative guideline or instruction to the agency field unit that defines the

extent of contamination at which FDA may regard food as adulterated. An action

level represents the limit at or above which FDA may take legal action to remove

products from the marketplace. Under the Food, Drug, and Cosmetic Act, FDA

also may set tolerances for unavoidably added poisonous or deleterious

substances, that is, substances that are either required in the production of food

or are otherwise unavoidable by good manufacturing practices. A tolerance is a

regulation that is established following formal rulemaking procedures; an action

level is a guideline or “instruction” and is not a formal regulation (Boyer et al.,

1991). 

FDA’s jurisdiction in setting action levels or tolerances is limited to contaminants

in food shipped and marketed in interstate commerce. Thus, the methodology

used by FDA in establishing action levels or tolerances is directed at determining

the health risks of chemical contaminants in fish and shellfish that are bought and

sold in interstate commerce rather than in locally harvested fish and shellfish

(Bolger et al., 1990). FDA action levels and tolerances are indicators of chemical

residue levels in fish and shellfish that should not be exceeded for the general

population who consume fish and shellfish typically purchased in supermarkets

or fish markets that sell products that are harvested from a wide geographic area,

including imported fish and shellfish products. However, the underlying assumptions used in the FDA methodology were never intended to be protective of

recreational, tribal, ethnic, and subsistence fishers who typically consume larger

quantities of fish than the general population and often harvest the fish and

shellfish they consume from the same local waterbodies repeatedly over many

years. If these local fishing and harvesting areas contain fish and shellfish with

elevated tissue levels of chemical contaminants, these individuals potentially

could have increased health risks associated with their consumption of the

contaminated fish and shellfish.

The following chemical contaminants discussed in this volume have FDA action

levels for their concentration in the edible portion of fish and shellfish: chlordane,

DDT, DDE, DDD, heptachlor epoxide, mercury, and mirex. FDA has not set an

action level for PCBs in fish but has established a tolerance in fish for this

chemical. Table 1-3 compares the FDA action levels and tolerance for these six

chemical contaminants with EPA’s recommended screening values (SVs) for

recreational and subsistence fishers calculated for these target analytes using the

EPA methodology.

The EPA SV for each chemical contaminant is defined as the concentration of the

chemical in fish tissue that is of potential public health concern and that is used

as a threshold value against which tissue residue levels of the contaminant in fish

and shellfish can be compared. The SV is calculated based on both the
1. INTRODUCTION

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noncarcinogenic and carcinogenic effects of the chemical contaminant, which are

discussed in detail in Section 5 of this volume. EPA recommends that the more

conservative of the calculated values derived from the noncarcinogenic rather

than the carcinogenic effects be used because it is more protective of the

consumer population (either recreational or subsistence fishers). As can be seen

in Table 1-3 for the recreational fisher SV, the EPA-recommended values typically

range from 2 to 120 times lower and are thus more protective than the

corresponding FDA action or tolerance level. This difference is even more striking

for subsistence fishers for whom the SVs are 20 to 997 times lower than the FDA

values.

EPA and FDA have agreed that the use of FDA Action Levels for the purpose of

making local advisory determinations is inappropriate. In letters to all states,

guidance documents, and annual conferences, this practice has been discouraged by EPA and FDA in favor of EPA’s risk-based approach to derive local fish

consumption advisories.

EPA has provided this guidance to be especially protective of recreational fishers

and subsistence fishers within the general U.S. population. EPA recognizes,

however, that Native American subsistence fishers are a unique subsistence

fisher population that needs to be considered separately. For Native American

subsistence fishers, eating fish is not simply a dietary choice that can be

completely eliminated if chemical contamination reaches unacceptable levels;

rather, eating fish is an integral part of their lifestyle and culture. This traditional

lifestyle is a living religion that includes values about environmental responsibility

and community health as taught by elders and tribal religious leaders (Harris and

Harper, 1977). Therefore, methods for balancing benefits and risks from eating

Table 1-3. Comparison of FDA Action Levels and Tolerances with EPA

Screening Values

Chemical contaminant

FDA

Action Levela

(ppm)

EPA SV for

Recreational Fishers

(ppm)

EPA SV for

Subsistence

Fishers (ppm)

Chlordane 0.3 0.114 0.014

Total DDT 5 0.117 0.014 

Dieldrin 0.3 2.50 x 10-3 3.07 x 10-4

Heptachlor epoxide 0.3 4.39 x 10-3 5.40 x 10-4

Mercury 1 .0 0.40 0.049 

Mirex 0.1 0.80 0.098 

FDA Tolerance

Level (ppm)

PCBs 2 0.02 2.45 x 10-3

a

U.S. FDA 1998.
1. INTRODUCTION

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contaminated fish must be evaluated differently than for the general fisher

population (see Section 5.1.3.2). 

To enhance the use of this guidance as a working document, EPA will issue

additional information and updates to users as appropriate. It is anticipated that

updates will include minor revisions such as the addition or deletion of chemicals

from the recommended list of target analytes, new screening values as new

toxicologic data become available, and new chemical analysis procedures for

some target analytes as they are developed. A new edition of this document will

be issued to include the addition of major new areas of guidance or when major

changes are made to the Agency’s risk assessment procedures.

EPA’s Office of Water realizes that adoption of these recommended methods

requires adequate funding. In practice, funding varies among states and resource

limitations will cause states to tailor their fish and shellfish contaminant monitoring

programs to meet their own needs. States must consider tradeoffs among the

various parameters when developing their fish contaminant monitoring programs.

These parameters include

• Total number of stations sampled

• Intensity of sampling at each site

• Number of chemical analyses and their cost

• Resources expended on data storage and analysis, QA and quality control

(QC), and sample archiving. 

Consideration of these tradeoffs will determine the number of sites sampled,

number of target analytes analyzed at each site, number of target species

collected, and number of replicate samples of each target species collected at

each site (Crawford and Luoma, 1993).

1.3 OBJECTIVES

The specific objectives of this manual are to

1. Recommend a tiered monitoring strategy designed to 

• Screen waterbodies (Tier 1) to identify those harvested sites where

chemical contaminant concentrations in the edible portions of fish and

shellfish exceed human consumption levels of potential concern

(screening values [SVs]). SVs for contaminants with carcinogenic effects

are calculated based on selection of an acceptable cancer risk level. SVs

for contaminants with noncarcinogenic effects are concentrations

determined to be without appreciable noncancer health risk. For a

contaminant with both carcinogenic and noncarcinogenic effects, EPA

recommends that the lower (more conservative) of these two calculated

SVs be used.
1. INTRODUCTION

1-14

• Conduct intensive followup sampling (Tier 2, Phase I) to determine the

magnitude of the contamination in edible portions of fish and shellfish

species commonly consumed by humans in waterbodies identified in the

screening process.

• Conduct intensive sampling at additional sites (Tier 2, Phase II) in a

waterbody where screening values were exceeded to determine the

geographic extent of contamination in various size classes of fish and

shellfish.

• Conduct intensive followup sampling in waterbodies where none of the 25

SVs are exceeded in order to establish areas of unrestricted fish

consumption or “green areas.”

2. Recommend target species and criteria for selecting additional species if the

recommended target species are not present at a site.

3. Recommend target analytes to be analyzed in fish and shellfish tissue and

criteria for selecting additional analytes.

4. Recommend risk-based procedures for calculating target analyte screening

values.

5. Recommend standard field procedures including 

• Site selection

• Sampling time

• Sample type and number of replicates

• Sample collection procedures including sampling equipment

• Field recordkeeping and chain of custody

• Sample processing, preservation, and shipping.

6. Recommend cost-effective, technically sound analytical methods and

associated QA and QC procedures, including identification of

• Analytical methods for target analytes with detection limits capable of

measuring tissue concentrations at or below SVs

• Sources of recommended certified reference materials 

• Federal agencies currently conducting QA interlaboratory comparison

programs.

7. Recommend procedures for data analysis and reporting of fish and shellfish

contaminant data.

8. Recommend QA and QC procedures for all phases of the monitoring program

and provide guidance for documenting QA and QC requirements in a QA plan

or in a combined work/QA project plan.
1. INTRODUCTION

1-15

1.4 RELATIONSHIP OF MANUAL TO OTHER GUIDANCE DOCUMENTS

This manual is the first in a series of four documents to be prepared by EPA’s

Office of Water as part of a Federal Assistance Plan to help states standardize

fish consumption advisories. This series of four documents—Guidance for

Assessing Chemical Contaminant Data for Use in Fish Advisories includes

• Volume 1: Fish Sampling and Analysis (EPA 823-R-93-002), published

August 1993; a second edition, published September 1995; and the current

third edition (EPA-823-B-00-007) to be published in November 2000.

• Volume 2: Risk Assessment and Fish Consumption Limits (EPA 823-B-94-

004), published June 1994; a second edition (EPA 823-B-97-009), published

in July 1997; and a third edition (EPA-823-B-00-008) to be published in

November 2000.

• Volume 3: Overview of Risk Management (EPA 823-B-96-006), published in

June 1996.

• Volume 4: Risk Communication (EPA 823-R-95-001), published March 1995.

This sampling and analysis manual is not intended to be an exhaustive guide to

all aspects of sampling, statistical design, development of risk-based screening

values, laboratory analyses, QA and QC considerations, data analysis, and

reporting for fish and shellfish contaminant monitoring programs. Key references

are provided in Section 10, Literature Cited, that detail various aspects of these

topics.

1.5 CONTENTS OF VOLUME 1

Figure 1-3 shows how Volume 1 fits into the overall guidance series and lists the

major categories of information provided. The first five sections discuss the

history of the EPA Fish and Wildlife Contamination Program, monitoring strategy,

including selection of target fish and shellfish species, selection of target analytes,

and calculation of screening values for all target analytes. Section 6 provides

guidance on field sampling and preservation procedures. Sections 7 and 8

provide guidance on laboratory procedures including sample handling and

analysis, and Section 9 discusses data analysis and reporting procedures. 

Appropriate QA and QC considerations are integral parts of each of the

recommended procedures. Section 10 is a compilation of all literature cited in

Sections 1 through 9 of this document. New information or revisions to existing

information contained in previous editions of this guidance document are briefly

described in Section 1.6.

Section 1 of this document reviews the historical development of this guidance

document series, describes the purpose and objectives of the Volume 1 manual,
1. INTRODUCTION

1-16

1. Introduction

2. Monitoring Strategy 2. Monitoring Strategy

3. Target Species

4. Target Analytes

5. Screening Values for

Target Analytes

6. Field Procedures

7. Laboratory Procedures I—

Sample Handling

8. Laboratory Procedures II—

Sample Analyses

9. Data Analysis and

Reporting

10. Literature Cited

Volume 3: Overview

of Risk Management

Volume 3: Overview

of Risk Management

Volume 1: Fish

Sampling and Analysis

Volume 1: Fish

Sampling and Analysis

Volume 2: Risk

Assessment and Fish

Consumption Limits

Volume 2: Risk

Assessment and Fish

Consumption Limits

Volume 4: Risk

Communication

Volume 4: Risk

Communication

Figure 1-3. Series summary: Guidance for assessing chemical

contamination data for use in fish advisories.
1. INTRODUCTION

1-17

outlines the relationship of the manual to the other three documents in the series,

describes the contents of the manual, and identifies new revisions made to the

guidance of this third edition. 

Section 2 outlines the recommended strategy for state fish and shellfish

contaminant monitoring programs. This strategy is designed to (1) routinely

screen waterbodies to identify those locations where chemical contaminants in

edible portions of fish and shellfish exceed human health screening values, (2)

sample more intensively those waterbodies where exceedances of these SVs

have been found in order to assess the magnitude and the geographic extent of

the contamination, and (3) identify those areas where chemical contaminant

concentrations are low and would allow states to designate areas where

unrestricted fish consumption may be permitted. 

Section 3 discusses the purpose of using target species and criteria for selection

of target species for both screening and intensive studies. Lists of recommended

target species are provided for inland fresh waters, Great Lakes waters, and

seven distinct estuarine and coastal marine regions of the United States.

Section 4 presents a list of recommended target analytes to be considered for

inclusion in screening and intensive studies, briefly discusses the original criteria

used in selecting these analytes, provides a summary of the toxicological

information available for each analyte as well as pertinent information on the

analyte’s detection in national and regional fish monitoring studies. 

Section 5 describes the new EPA risk-based procedure for calculating screening

values for target analytes using (1) an adult body weight of 70 kg, (2) a lifetime

exposure of 70 years, and (3) new consumption rate default values for both the

general population and recreational fishers (17.5 g/d) and subsistence fishers

(142.4 g/d). The last part of this section describes how to compare these new

SVs against results obtained in fish tissue residue analysis.

Section 6 recommends field procedures to be followed from the time fish or

shellfish samples are collected until they are delivered to the laboratory for

processing and analysis. Guidance is provided on site selection and sample

collection procedures; the guidance addresses material and equipment

requirements, time of sampling, size of animals to be collected, sample type, and

number of samples. Sample identification, handling, preservation, shipping, and

storage procedures are also described.

Section 7 describes recommended laboratory procedures for sample handling

including: sample measurements, sample processing procedures, and sample

preservation and storage procedures. 

Section 8 presents recommended laboratory procedures for sample analyses,

including cost-effective analytical methods and associated QC procedures; and

information on sources of certified reference materials; recommended analytical
1. INTRODUCTION

1-18

techniques for target analytes, including revised detection and quantitation limits;

information on the per-sample cost of chemical analysis for each target analyte;

and information on federal agencies currently conducting interlaboratory

comparison programs.

Section 9 includes procedures for data analysis to determine the need for additional monitoring and risk assessment and for data reporting. 

Supporting documentation for this guidance is provided in Section 10, Literature

Cited and in Appendixes A through N. 

1.6 NEW INFORMATION AND REVISIONS TO VOLUME 1

This 3rd edition of Volume 1 contains newly prepared material as well as major

updates and revisions to existing information. A brief summary of major additions

and revisions is provided below.

Section 1

• New information is presented on the NLFWA database, including the 5-year

trend in the total number of advisories issued nationwide, the number of

advisories issued for five major pollutants of concern, and the issuance of

increasing numbers of statewide advisories for freshwater lakes and/or rivers

and coastal marine areas.

• Additional information describes the flexibility that is built into the EPA

methodology, which allows the method to be used to meet a wide variety of

state or tribal study needs within budgetary constraints.

• Clarification of the FDA methodology is provided emphasizing the

inappropriateness of the method and reasons states should adopt and use the

EPA methodology when issuing fish consumption advisories to protect their

recreational and subsistence fishers. 

Section 2

• Updated information is presented in Table 2-1 to be consistent with monitoring

design and risk assumptions used in this 3rd edition.

• New discussion of the criteria states may use to identify green areas where

chemical contaminant concentrations are at or below the screening values for

recreational or subsistence fishers is introduced with more detailed

information provided in Appendix B.
1. INTRODUCTION

1-19

Section 3

• Several tables, including Tables 3-7 and 3-19, were updated to include new

information from the 1998 NLFWA database on the number of states that

have issued fish advisories for freshwater and marine species.

• Table 3-9 was updated and associated narrative text was revised to include

information on studies using turtles as biomonitors of environmental

contaminants.

Section 4

• Information on the environmental sources, toxicology, and the number of fish

advisories issued in 1998 for each of the 25 target analytes was updated.

• New information is included on the range in concentrations of each

contaminant detected in the FWS National Contaminant Biomonitoring

Program and the EPA National Study of Chemical Residues in Fish as well as

information on more recent regional studies. 

• A procedure is described for the selection and prioritization of target analytes

for analysis predicated on a watershed-based approach that takes into

consideration land use categories, as well as geological characteristics,

regional differences, national fish advisory trends, and monitoring and analysis

costs.

• Additional guidance is presented on organophosphate pesticides and when

and under what situations to monitor fish tissues for these compounds.

• A clarification is provided of the recommendation for selection of target

species, especially bivalve molluscs and/or crustaceans when PAH

contamination is suspected.

• A new discussion is provided to reflect the Agency’s position on using Aroclor

and congener analysis for calculating total PCB concentration.

• A new discussion is provided for determining the TEQ value for dioxins, which

are now defined as including the 17 2,3,7,8 congeners of dioxin and 2,3,7,8

congeners of dibenzofuran, and the 12 coplanar PCBs with dioxin-like

properties based on recent guidance from the World Health Organization (Van

den Berg et al., 1998).

• Several tables, including Tables 4-1, 4-2, 4-7, and 4-9 were revised with new

information. Tables 4-3, 4-4, 4-5, 4-6, and 4-8 are new to the document.

• All of the toxicological information was revised in light of the most current

information concerning each target analyte.
1. INTRODUCTION

1-20

Section 5

• Revisions were made describing major changes in the assumptions used in

the risk assessment equations to calculate screening values including use of

default consumption rates of 17.5 g/d for the general population and recreational fishers and 142.4 g/d for subsistence fishers based on more recent

information from the 1994 to 1996 Continuing Survey of Food Intake by

Individuals study conducted by the U.S. Department of Agriculture.

• Additional guidance is provided on how states should handle the interpretation

and risk assessment of chemicals that have detection limits higher than the

risk-based screening values.

• Tables 5-1, 5-3, 5-4, and 5-5 were revised to reflect changes in consumption

rates. Screening values shown in Tables 5-3 and 5-4 were developed using

the new consumption rates as well as the most recent RfD and cancer slope

factors available. 

• Additional information is provided on Native American subsistence fishers, and

Table 5-2 was added to summarize several recent studies on Native American

fish consumption rates.

• Additional guidance is provided on how states should deal with interpreting

analytical results in cases where the screening value is lower than the

detection limit for a particular analyte.

• New guidance is provided on determining total PCBs by summary Aroclor

equivalents or PCB congeners.

• New information from the World Health Organization (Van den Berg et al.,

1998) is included in Table 5-6 showing the most recent Toxic Equivalency

Factors (TEF) for the 2,3,7,8-substituted dioxins, dibenzofurans, and the

12 coplanar PCBs.

Section 6

• Additional information is provided on the statistical implications associated

with deviations from the recommended sampling design, including the use of

unequal numbers of fish per composite, sizes of fish exceeding the size range

recommendations for composites, and the use of unequal numbers of

replicate samples across sampling sites.

• Clarification is provided on the recommended number of fish that should make

up a composite sample.
1. INTRODUCTION

1-21

• More explicit information is provided regarding exceedances of screening

values and the statistical basis for issuing a new advisory or rescinding an

existing advisory.

 

• Discussion is provided on the number of samples necessary to characterize

different waterbody types and sizes of waterbodies with consideration given

to the home range and mobility of the target species.

• How regional data should be used in the risk assessment process to address

statewide advisories is discussed.

• Additional guidance is provided on how sample type selection should be

based on the study objectives as well as on the sample type consumed by the

target population.

• Clarification is provided as to EPA’s position on the use of dead, lacerated, or

mutilated fish for human health risk assessments.

• New information is provided on U.S. Fish and Wildlife Service and National

Marine Fisheries permit requirements in situations where concerns exist about

the impact of sampling for the target species in areas inhabited by threatened

or endangered species.

• Revisions were made in recordkeeping for field sampling associated with use

of the Year 2000 compliant format (YYYYMMDD) for sampling date

information. 

Section 7

• Revisions were made in recordkeeping forms to initiate use of the Year 2000

compliant format for the date of sampling and analysis procedures.

Section 8

• Updated information is included in Tables 8-1 through 8-5.

• Updated information is provided on the EPA Environmental Monitoring

Methods Index System (EMMI).

• Revised information is provided in Section 8.3.3.8.1 concerning round-robin

analysis interlaboratory comparison programs.

Section 9

• New information is included on the National Tissue Residue Data Repository,

now housed within the NLFWA database.
1. INTRODUCTION

1-22

• Recommended data reporting requirements were updated (Figure 9-1) to

include Year 2000 compliant format.

• Detailed information is provided on the Internet-based data entry facility

contained within the NLFWA database that can accept fish contaminant

residue data to support state fish advisories.

• An example of the new data tables (Figure 9-2) currently used in the fish

tissue residue data repository is provided.

Section 10

• Literature citations were revised to include all new references cited in

Sections 1 through 9.

Appendixes:

• The following appendixes were revised or added:

A - EPA 1993 Fish Contamination Workgroup Members

B - Screening Values for Defining Green Areas

D - Fish and Shellfish Species for Which State Consumption Advisories Have

Been Issued

F - Pesticide and Herbicides Recommended as Target Analytes

G - Target Analyte Dose-Response Variables and Associated Information

I - Quality Assurance and Quality Control Guidance

M - Sources of Reference Materials 
2. MONITORING STRATEGY

2-1

SECTION 2

MONITORING STRATEGY

The objective of this section is to describe the strategy recommended by the EPA

Office of Water for use by states in their fish and shellfish contaminant monitoring

programs. A two-tiered strategy is recommended as the most cost-effective

approach for State contaminant monitoring programs to obtain data necessary to

evaluate the need to issue fish or shellfish consumption advisories. This

monitoring strategy is shown schematically in Figure 2-1 and consists of

• Tier 1—Screening studies of a large number of sites for chemical

contamination where sport, subsistence, and/or commercial fishing is

conducted. This screening will help states identify those sites where

concentrations of chemical contaminants in edible portions of commonly

consumed fish and shellfish indicate the potential for significant health risks

to human consumers.

• Tier 2—Two-phase intensive studies of problem areas identified in

screening studies to determine the magnitude of contamination in edible

portions of commonly consumed fish and shellfish species (Phase I), to

determine size-specific levels of contamination, and to assess the geographic

extent of the contamination (Phase II).

One key objective in the recommendation of this approach is to improve the data

used by states for issuing fish and shellfish consumption advisories. Other

specific aims of the recommended strategy are

• To ensure that resources for fish contaminant monitoring programs are

allocated in the most cost-effective way. By limiting the number of sites

targeted for intensive studies, as well as the number of target analytes at each

intensive sampling site, screening studies help to reduce overall program

costs while still allowing public health protection objectives to be met.

• To ensure that sampling data are appropriate for developing risk-based

consumption advisories.

• To ensure that sampling data are appropriate for determining contaminant

concentrations in various size (age) classes of each target species so that

states can give size-specific advice on contaminant concentrations (as

appropriate).
2. MONITORING STRATEGY

2-2
2. MONITORING STRATEGY

2-3Figure 2-1. Recommended strategy for state fish and shellfish contaminant monitoring programs.
2. MONITORING STRATEGY

2-4

• To ensure that sampling designs are appropriate to allow statistical hypothesis

testing. Such sampling designs permit the use of statistical tests to detect a

difference between the average tissue contaminant concentration at a site and

the human health screening value for any analyte.

The following elements must be considered when planning either screening

studies or more intensive followup sampling studies:

• Study objective

• Target species (and size classes)

• Target analytes

• Target analyte screening values

• Sampling locations

• Sampling times

• Sample type

• Sample replicates

• Sample analysis

• Data analysis and reporting.

Detailed guidance for each of these elements, for screening studies (Tier 1) and

for both Phase I and Phase II of intensive studies (Tier 2), is provided in this

document. The key elements of the monitoring strategy are summarized in

Table 2-1, with reference to the section number of this document where each

element is discussed.

2.1 SCREENING STUDIES (TIER 1)

The primary aim of screening studies is to identify frequently fished sites where

concentrations of chemical contaminants in edible fish and shellfish composite

samples exceed specified human health screening values and thus require more

intensive followup sampling. Ideally, screening studies should include all waterbodies where commercial, recreational, or subsistence fishing is practiced;

specific sampling sites should include areas where various types of fishing are

conducted routinely (e.g., from a pier, from shore, or from private and commercial

boats), thereby exposing a significant number of individuals to potentially adverse

health effects. Composites of skin-on fillets (except for catfish and other scaleless

species, which are usually prepared as skin-off fillets) and edible portions of

shellfish are recommended for contaminant analyses in screening studies to

provide conservative estimates of typical exposures for the general population.

If consumers remove the skin and fatty areas from a fish before preparing it for

eating, exposures to some contaminants can be reduced (see U.S. EPA, 2000a,

Appendix C of Volume 2 of this guidance document series).

Note: If the target population of consumers includes primarily ethnic or

subsistence fishers who consume the whole fish or tissues of the fish not typically

consumed by the general population, state monitoring programs should include

the fish sample type associated with the target consumers’ dietary and/or culinary

preference (see Section 6.1.1.6, Sample Type, for additional information.)
2. MONITORING STRATEGY

2-5See notes at end of table. (continued)

Table 2-1. Recommended Strategy for State Fish and Shellfish Contaminant Monitoring Programs

Program element Tier 1 Screening study Tier 2 Intensive study (Phase I) Tier 2 Intensive study (Phase II)

Objective

(see Section 2)

Identify frequently fished sites where commonlyconsumed fish and shellfish target species arecontaminated and may pose potential humanhealth risk.

Assess and verify magnitude of tissuecontamination at screening site forcommonly consumed target species.

Assess geographic extent ofcontamination in selected size classesof commonly consumed target species.

Target species and

size classes(see Sections 3and 6)

Select target species from commonlyconsumed species using the followingadditional criteria: known to bioaccumulate highconcentrations of contaminants and distributedover a wide geographic area.

Resample target species at sites wherethey were found to be contaminated inscreening study.

Resample at additional sites in thewaterbody 3 size classes of the targetspecies found to be contaminated inPhase I study.

Recommended types of target species:Inland fresh 1 bottom-feederwaters: 1 predatorGreat Lakes: 1 bottom-feeder

1 predator

Estuarine/ 1 shellfish andmarine: 1 fish species

or

2 fish species (one species should bebottom-feeder).

OPTIONAL: If resources are limited and astate cannot conduct Tier 2 intensive studies,the state may find it more cost-effective tocollect additional samples during the Tier 1screening study. States may collect (1) one

composite sample of each of three size classesfor each target species, (2) replicate compositesamples for each target species, or (3) replicatecomposite samples of each of three sizeclasses for each target species.

OPTIONAL: If resources are limitedand a state cannot conduct Tier 2,Phase II, intensive studies, the statemay find it more cost-effective to collectadditional samples during the Tier 2,Phase I, intensive study. States maycollect replicate composite samples ofthree size classes of the target speciesfound to be contaminated to assesssize-specific contaminant concentrations. Other commonly consumedtarget species may also be sampled ifresources allow.

OPTIONAL: If resources allow, selectadditional commonly consumed targetspecies using same criteria as in PhaseI study.
2. MONITORING STRATEGY

2-6

Table 2-1. (continued)

Program element Tier 1 Screening study Tier 2 Intensive study (Phase I) Tier 2 Intensive study (Phase II)

See notes at end of table. (continued)

Target analytes(see Section 4)

Consider all target analytes listed in Table 4-1for analysis but prioritize the 25 target analytesbased on water and sediment sampling results,land use within the watershed, geographiccharacteristics, regional and national advisorytrends and analytical costs. Include additionalsite-specific target analytes as appropriatebased on current or historic data.

Analyze only for those target analytesfrom Tier 1 screening study thatexceeded SVs.

Analyze only for those target analytesfrom Tier 2, Phase I, study thatexceeded SVs.

Screening values

(see Section 5)

Calculate SVs using oral RfDs for noncarcinogens and using oral slope factors and anappropriate risk level (10-4 to 10-7) for carcinogens, for adults consuming 17.5 g/d and

142.4 g/d of fish and shellfish (default values)or based on site-specific dietary data.Note: In this guidance document, EPA’s Officeof Water used 17.5 g/d (for recreational fishers)and 142.4 g/d (for subsistence fishers)consumption rates, 70-kg adult body weight,and, for carcinogens, used a 10-5 risk level,

70-year exposure, and assumed no loss ofcontaminants during preparation or cooking. States may use other SVs for site-specificexposure scenarios by adjusting values forconsumption rate, body weight, risk level,exposure period, and contaminant loss duringpreparation or cooking.

Use same SVs as in screening study. Use same SVs as in screening study.

Sampling sites

(see Section 6)

Sample target species at sites in each harvestarea that have a high probability of contamination and at presumed clean sites or given areasas resources allow (see Appendix A).

Sample target species at each siteidentified in the screening study wherefish/shellfish tissue concentrationsexceed SVs to assess the magnitude ofcontamination.

Sample at additional sites in the harvestarea 3 size classes of the target speciesfound to be contaminated in Phase Istudy to assess the geographic extent ofthe contamination in the waterbody.
2. MONITORING STRATEGY

2-7

Table 2-1. (continued)

Program element Tier 1 Screening study Tier 2 Intensive study (Phase I) Tier 2 Intensive study (Phase II)

See notes at end of table. (continued)

Sampling times(see Section 6)

Sample during legal harvest season whentarget species are most available toconsumers. Ideally, sampling time should notinclude the spawning period for target speciesunless the target species can be legallyharvested during this period.

Same as screening study. Same as screening study.

Sample type

(see Sections 6and 7)

Collect composite fillet samples (skin on, bellyflap included) for each target fish species andcomposite samples of edible portions of targetshellfish species. The exceptions to the "skinon, belly flap included" recommendation is touse skin-off fillets for catfish and otherscaleless species.

Same as screening study. Same as screening study but collect

composite samples for three sizeclasses of each target species asappropriate.

OPTIONAL: States may use individual fishsamples, whole fish, or other sample types, ifnecessary, to improve exposure estimates oflocal fish-, shellfish-, or turtle-consumingpopulations. Sample type should reflect dietaryand fish preparation methods of the targetpopulation of concern.

Same as screening study. Same as screening study.

Sample replicates

(see Section 6)

Collect one composite sample for each targetspecies. Collection of replicate composite

samples is encouraged but is optional. If

resources allow, collect a minimum of onereplicate composite sample for each targetspecies at 10% of the screening sites for QC.

Collect replicate composites for eachtarget species at each Phase I site.

Collect replicate composites of threesize classes for each target species ateach Phase II site.

Sample analysis

(see Section 8)

Use standardized and quantitative analyticalmethods with limits of detection adequate toallow reliable quantitation of selected targetanalytes at or below SVs.

Use same analytical methods as inscreening study.

Use same analytical methods as inscreening study.
2. MONITORING STRATEGY

2-8

Table 2-1. (continued)

Program element Tier 1 Screening study Tier 2 Intensive study (Phase I) Tier 2 Intensive study (Phase II)

See notes at end of table. (continued)

Data analysis andreporting(see Sections 6,7, 8, and 9)

For each target species, compare targetanalyte concentrations of composite samplewith SVs to determine which sites requireTier 2, Phase I, intensive study.

For each target species, comparetarget analyte arithmetic meanconcentrations of replicate compositesamples with SVs to determine whichsites require Phase II intensive study. If resources are insufficient to conductPhase II intensive study, conduct a riskassessment and assess the need forissuing a preliminary fish or shellfishconsumption advisory.

For each of three size classes withineach target species, compare targetanalyte arithmetic mean concentrationsof replicate composite samples at eachPhase II site with SVs to determinegeographic extent of fish or shellfishcontamination. Assess the need forissuing a final fish or shellfishconsumption advisory.

Data analysis and

reporting(see Sections 6,7, 8, and 9)(continued)

The following information should be reportedfor each target species at each site:

The following information should bereported for each target species at eachsite:

The following information should bereported for each of three size classeswithin each target species at each site:

• Site location (e.g., sample site name, waterbody name, type of waterbody, and

latitude/longitude)

• Same as screening study. • Same as screening study.

• Scientific and common name of target 

species

• Same as screening study • Same as screening study

• Sampling date and time• Sampling gear type used• Sampling depth• Number of QC replicates (optional)• Number of individual organisms used in the

composite sample and in the QC replicatecomposite sample if applicable

• Same as screening study• Same as screening study• Sampling depth• Number of replicates• Number of individual organisms

used in each replicate compositesample

• Same as screening study• Same as screening study• Sampling depth• Same as Phase I study• Same as Phase I study
2. MONITORING STRATEGY

2-9

Table 2-1. (continued)

Program element Tier 1 Screening study Tier 2 Intensive study (Phase I) Tier 2 Intensive study (Phase II)

See notes at end of table. (continued)

• Predominant characteristics of specimens

used in the composite sample and in theQC replicate if applicable (e.g., life stage,age, sex, total length or body size) anddescription of fish fillet or edible parts ofshellfish (tissue type) used

• Predominant characteristics of

specimens used in each replicatecomposite sample (e.g., life stage,age, sex, total length or body size)and description of fish fillet or edibleparts of shellfish (tissue type) used

• Same as Phase I study

• Analytical methods used (including a

method for lipid analysis) and method detection and quantitation limits for eachtarget analyte.

• Same as screening study • Same as screening study

Data analysis and

reporting(see Sections 6,7, 8, and 9)(continued)

• Sample cleanup procedures• Data qualifiers• Percent lipid in each composite sample.

• Same as screening study.• Same as screening study.• Same as screening study.

• Same as screening study.• Same as screening study.• Same as screening study.

• For each target analyte: • For each target analyte: • For each target analyte:

– Total wet weight of composite sample

(g) used in analysis

– Total wet weight of each

replicate composite sample (g)used in analysis

– Same as Phase I study

– Measured concentration (wet weight) in

composite sample including units ofmeasurement for target analyte

– Measured concentration (wet

weight) in each replicatecomposite sample and units ofmeasurement for target analyte

– Same as Phase I study

– Measured concentration (wet weight) in

the QC replicate, if applicable

– Range of concentrations (wet

weight) for each set of replicatecomposite samples

– Same as Phase I study

– Mean (arithmetic) concentration

(wet weight) for each set ofreplicate composite samples

– Same as Phase I study

– Standard deviation of mean

concentration (wet weight)

– Same as Phase I study
2. MONITORING STRATEGY

2-10

Table 2-1. (continued)

Program element Tier 1 Screening study Tier 2 Intensive study (Phase I) Tier 2 Intensive study (Phase II)

– Evaluation of laboratory performance

(i.e., description of all QA and QCsamples associated with the sample(s)and results of all QA and QC analyses)

– Same as screening study – Same as screening study

– Comparison of measured concentration

of composite sample with SV and clearindication of whether SV was exceeded

– Comparison of target analyte

arithmetic mean concentration ofreplicate composite sampleswith SV using hypothesis testingand clear indication of whetherthe SV was exceeded

– Same as Phase I study

QA = Quality assurance. RfDs = Reference doses.QC = Quality control. SVs = Screening values.
2. MONITORING STRATEGY

2-11

Because the sampling sites in screening studies are focused primarily on the most

likely problem areas and the numbers of commonly consumed target species and

samples collected are limited, relatively little detailed information is obtained on

the magnitude and geographic extent of contamination in a wide variety of

harvestable fish and shellfish species of concern to consumers. More information

is obtained through additional intensive followup studies (Tier 2, Phases I and II)

conducted at potentially contaminated sites identified in screening studies. 

Although the EPA Office of Water recommends that screening study results not

be used as the sole basis for conducting a risk assessment, EPA recognizes that

this practice may be unavoidable if monitoring resources are limited or if the state

must issue an advisory based on detection of elevated concentrations in one

composite sample. States have several options for collecting samples during the

Tier 1 screening study (see Figure 2-1), which can provide additional information

on contamination without necessitating additional field monitoring expenditures

as part of the Tier 2 intensive studies.

The following assumptions are made in this guidance document for sampling fish

and shellfish and for calculating human health SVs for recreational and

subsistence fishers:

• Use of commonly consumed target species that are dominant in the catch and

have high bioaccumulation potential (see Section 3, Target Species)

• Use of fish fillets (with skin on and belly flap tissue included) for scaled finfish

species, use of skinless fillets for scaleless finfish species, and use of edible

portions of shellfish (see Section 6.1.1.6, Sample Type)

• Use of fish and shellfish above legal size to maximum size in the target species

• Use of a 10-5 risk level, a human body weight of 70 kg (average adult), a

consumption rate of 17.5 g/d for recreational fishers and 142.4 g/d for

subsistence fishers, and a 70-yr lifetime exposure period to calculate SVs for

carcinogens.

• Use of a human body weight of 70 kg (average adult) and a consumption rate

of 17.5 g/d for recreational fishers and 142.4 g/d for subsistence fishers to

calculate SVs for noncarcinogens (see Section 5, Screening Values for Target

Analytes).

• Use of no contaminant loss during preparation and cooking or from incomplete

absorption in the intestines.

For certain site-specific situations, states may wish to use one or more of the

following exposure assumptions to protect the health of high-end fish consumers

such as subsistence fishers at potentially greater risk:
2. MONITORING STRATEGY

2-12

• Use of commonly consumed target species that are dominant in the catch and

have the highest bioaccumulation potential

• Use of whole fish or whole body of shellfish (excluding shell of bivalves), which

may provide a better estimate of contaminant exposures in ethnic or Native

American subsistence populations that consume whole fish or shellfish

• Use of the largest (oldest) individuals in the target species to represent the

highest likely exposure levels

• Use of a 10-6 or 10-7 risk level, body weights less than 70 kg for women and

children, site-specific consumption rates for sport fishers or for subsistence

fishers or other consumption rates based on dietary studies of local fishconsuming populations, and a 70-yr exposure period to calculate SVs for

carcinogens. Note: EPA has reviewed national data on the consumption

rate for sport and subsistence fishers and the recommended default values for

these populations are 17.5 and 142.4 g/d, respectively (USDA/ARS, 1998; U.S.

EPA, 2000c).

• Use of body weights less than 70 kg for women and children and site-specific

consumption rates for sport fishers or for subsistence fishers or other

consumption rates based on dietary studies of local fish-consuming

populations to calculate SVs for noncarcinogens. Note: EPA has reviewed

national data on the consumption rate for sport and subsistence fishers and

the recommended default values for these populations are 17.5 and 142.4 g/d,

respectively (USDA/ARS, 1998; U.S. EPA, 2000c).

There are additional aspects of the screening study design that states should

review because they affect the statistical analysis and interpretation of the data.

These include

• Use of composite samples, which results in loss of information on the

distribution of contaminant concentrations in the individual sampled fish and

shellfish. Maximum contaminant concentrations in individual sampled fish,

which can be used as an indicator of potentially harmful levels of contamination

(U.S. EPA, 1989d), are not available when composite sampling is used.

• Use of a single sample per screening site for each target species, which

precludes estimating the variability of the contamination level at that site and,

consequently, of conducting valid statistical comparisons to the target analyte

SVs.

• Uncertainty factors affecting the numerical calculation of quantitative health

risk information (i.e., references doses and cancer slope factors) as well as

human health SVs.
2. MONITORING STRATEGY

2-13

The use of composite samples is often the most cost-effective method for estimating average tissue concentrations of analytes in target species populations to

assess chronic human health risks. However, there are some situations in which

individual sampling can be more appropriate from both ecological and risk

assessment perspectives. Individual sampling provides a direct measure of the

range and variability of contaminant levels in target fish populations. Information

on maximum contaminant concentrations in individual fish is useful in evaluating

acute human health risks. Estimates of the variability of contaminant levels

among individual fish can be used to ensure that studies meet desired statistical

objectives. For example, the population variance of a contaminant can be used

to estimate the sample size needed to detect statistically significant differences

in contaminant screening values compared to the mean contaminant concentration. Finally, the analysis of individual samples may be desirable, or necessary,

when the objective is to minimize the impacts of sampling on certain vulnerable

target populations, such as predators in headwater streams and aquatic turtles,

and in cases where the cost of collecting enough individuals for a composite

sample is excessive. For states that wish to consider use of individual sampling

during either the screening or intensive studies, additional information on

collecting and analyzing individual samples is provided in Appendix C. States

should consider the potential effects of these study design features when

evaluating screening study results.

Note: As part of screening studies, states may wish to issue information not only

on restricting or avoiding consumption of certain species from certain waterbodies, but on promoting unrestricted fish consumption in those waterbodies

where the levels of contamination are below the SVs for all 25 of the target

analytes. Waterbodies in which target analyte concentrations (see Section 5) are

below the selected target analyte SVs are known as “green areas” where states

can promote fish consumption to specified fisher populations. Guidance to assist

states in designating these safe or green areas is provided in detail in Appendix B.

2.2 INTENSIVE STUDIES (TIER 2)

The primary aims of intensive studies are to assess the magnitude of tissue

contamination at screening sites, to determine the size class or classes of fish

within a target species whose contaminant concentrations exceed the SVs, and

to assess the geographic extent of the contamination for the target species in the

waterbody under investigation. With respect to the design of intensive studies,

EPA recommends a sampling strategy that may not be feasible for some sitespecific environments. Specifically, EPA recognizes that some waterbodies

cannot sustain the same intensity of sampling (i.e., number of replicate composite

samples per site and number of individuals per composite sample) that others

(i.e., those used for commercial harvesting) can sustain. In such cases, state

fisheries personnel may consider modifying the sampling strategy (e.g., analyzing

individual fish) for intensive studies to protect the fishery resource. Although one

strategy cannot cover all situations, these sampling guidelines are reasonable for

the majority of environmental conditions, are scientifically defensible, and provide
2. MONITORING STRATEGY

2-14

information that can be used to assess the risk to public health. Regardless of the

final study design and protocol chosen for a fish contaminant monitoring program,

state fisheries, environmental, and health personnel should always evaluate and

document the procedures used to ensure that results obtained meet state

objectives for protecting human health.

The allocation of limited funds to screening studies or to intensive studies should

always be guided by the goal of conducting adequate sampling of state fish and

shellfish resources to ensure the protection of public health. The amount of

sampling that can be performed by a state will be determined by available

economic resources. Ideally, state agencies will allocate funds for screening as

many sites as is deemed necessary while reserving adequate resources to

conduct subsequent intensive studies at sites where excessive fish tissue

contamination is detected. State environmental and health personnel should use

all information collected in both screening and intensive studies to (1) conduct a

risk assessment to determine whether the issuance of an advisory is warranted,

(2) use risk management to determine the nature and extent of the advisory, and

then (3) effectively communicate this risk to the fish-consuming public. Additional

information on risk assessment, risk management, and risk communication

procedures will be provided in subsequent volumes in this series.
3. TARGET SPECIES

3-1

SECTION 3

TARGET SPECIES

The primary objectives of this section are to: (1) discuss the purpose of using

target species, (2) describe the criteria used by the 1993 EPA Fish Contaminant

Workgroup to select target species, and (3) provide lists of recommended target

species. Target species recommended for freshwater and estuarine/marine

ecosystems are discussed in Sections 3.3 and 3.4, respectively.

3.1 PURPOSE OF USING TARGET SPECIES

The use of target species allows comparison of fish, shellfish, and turtle tissue

contaminant monitoring data among sites over a wide geographic area.

Differences in habitat, food preferences, and rate of contaminant uptake among

various fish, shellfish, and turtle species make comparison of contaminant

monitoring results within a state or among states difficult unless the contaminant

data are from the same species. It is virtually impossible to sample the same

species at every site, within a state or region or nationally, due to the varying

geographic distributions and environmental requirements of each species.

However, a limited number of species can be identified that are distributed widely

enough to allow for collection and comparison of contaminant data from many

sites.

Three aims are achieved by using target species in screening studies. First,

states can cost-effectively compare contaminant concentrations in their state

waters and then prioritize sites where tissue contaminants exceed human health

screening values. In this way, limited monitoring resources can be used to

conduct intensive studies at sites exhibiting the highest degree of tissue

contamination in screening studies. By resampling target species used in the

screening study in Phase I intensive studies and sampling additional size classes

and additional target species in Phase II intensive studies as resources allow,

states can assess the magnitude and geographic extent of contamination in

species of commercial, recreational, or subsistence value. Second, the use of

common target species among states allows for more reliable comparison of

sampling information. Such information allows states to design and evaluate their

own contaminant monitoring programs more efficiently, which should further

minimize overall monitoring costs. For example, monitoring by one state of fish

tissue contamination levels in the upper reaches of a particular river can provide

useful information to an adjacent state on tissue contamination levels that might

be anticipated in the same target species at sampling sites downstream. Third,

the use of a select group of target fish, shellfish, and freshwater turtle species will

allow for the development of a national database for tracking the magnitude and
3. TARGET SPECIES

3-2

geographic extent of pollutant contamination in these target species nationwide

and will permit analyses of trends in fish, shellfish, and turtle contamination over

time.

3.2 CRITERIA FOR SELECTING TARGET SPECIES

The appropriate choice of target species is a key element of any chemical

contaminant monitoring program. Criteria for selecting target species used in the

following national fish and shellfish contaminant monitoring programs were

reviewed by the 1993 EPA Fish Contaminant Workgroup to assess their

applicability for use in selecting target species for state fish contaminant

monitoring programs:

• National Study of Chemical Residues in Fish (U.S. EPA)

• National Dioxin Study (U.S. EPA)

• 301(h) Monitoring Program (U.S. EPA)

• National Pesticide Monitoring Program (U.S. FWS)

• National Contaminant Biomonitoring Program (U.S. FWS)

• National Status and Trends Program (NOAA).

• National Water Quality Assessment Program (USGS).

The criteria used to select target species in many of these programs are similar

although the priority given each criterion may vary depending on program aims.

According to the 1993 EPA Fish Contaminant Workgroup, the most important

criterion for selecting target fish, shellfish, and turtle species for state contaminant

monitoring programs assessing human consumption concerns was that the

species were commonly consumed in the study area and were of commercial,

recreational, or subsistence fishing value. Two other criteria of major importance

are that the species have the potential to bioaccumulate high concentrations of

chemical contaminants and have a wide geographic distribution. EPA

recommends that states use the same criteria to select species for both screening

and intensive site-specific studies. 

In addition to the three primary criteria for target species selection, it is also

important that the target species be easy to identify taxonomically because there

are significant species-specific differences in bioaccumulation potential. Because

many closely related species can be similar in appearance, reliable taxonomic

identification is essential to prevent mixing of closely related species with the

target species. Note: Under no circumstance should individuals of more than

one species be mixed to create a composite sample (U.S. EPA, 1991e). It is also

both practical and cost-effective to sample target species that are abundant, easy

to capture, and large enough to provide adequate tissue samples for chemical

analyses.
3. TARGET SPECIES

3-3

It cannot be overemphasized that final selection of target species will require the

expertise of state fisheries biologists with knowledge of local species that best

meet the selection criteria and knowledge of local human consumption patterns.

Although, ideally, all fish, shellfish, or turtle species consumed from a given

waterbody by the local population should be monitored, resource constraints may

dictate that only a few of the most frequently consumed species be sampled.

In the next two sections, lists of recommended target species are provided for

freshwater ecosystems (inland fresh waters and the Great Lakes) and

estuarine/marine ecosystems (Atlantic, Gulf, and Pacific waters), and the methods

used to develop each list are discussed.

3.3 FRESHWATER TARGET SPECIES

As part of the two-tiered sampling strategy proposed for state fish contaminant

monitoring programs, EPA recommends that states collect one bottom-feeding

fish species and one predator fish species at each freshwater screening study

site. Some suggested target species for use in state fish contaminant monitoring

programs are shown in Table 3-1 for inland fresh waters and in Table 3-2 for

Great Lakes waters.

The lists of target species recommended by the 1993 EPA Fish Contaminant

Workgroup for freshwater ecosystems were developed based on a review of

species used in the following national monitoring programs:

• National Study of Chemical Residues in Fish (U.S. EPA)

• National Dioxin Study (U.S. EPA)

• National Pesticide Monitoring Program (U.S. FWS)

• National Contaminant Biomonitoring Program (U.S. FWS)

• National Water Quality Assessment Program (USGS)

and on a review of fish species cited in state fish consumption advisories or bans

(RTI, 1993). Separate target species lists were developed for inland fresh waters

(Table 3-1) and Great Lakes waters (Table 3-2) because of the distinct ecological

characteristics of these waters and their fisheries. Each target species list has

been reviewed by regional and state fisheries experts.

Use of two distinct ecological groups of finfish (i.e., bottom-feeders and predators)

as target species in freshwater systems is recommended. This permits

monitoring of a wide variety of habitats, feeding strategies, and physiological

factors that might result in differences in bioaccumulation of contaminants.

Bottom-feeding species may accumulate high contaminant concentrations from

direct physical contact with contaminated sediment and/or by consuming benthic

invertebrates and epibenthic organisms that live in contaminated sediment.

Predator species are also good indicators of persistent pollutants (e.g., mercury

or DDT and its metabolites) that may be biomagnified through several trophic

levels of the food web. Species used in several federal programs to assess the
3. TARGET SPECIES

3-4

Table 3-1. Recommended Target Species for Inland Fresh Waters

Family name Common name Scientific name

Percichthyidae White bass Morone chrysops

Centrarchidae Largemouth bass

Smallmouth bass

Black crappie

White crappie

Micropterus salmoides

Micropterus dolomieui

Pomoxis nigromaculatus

Pomoxis annularis

Percidae Walleye

Yellow perch

Stizostedion vitreum

Perca flavescens

Cyprinidae Common carp Cyprinus carpio

Catostomidae White sucker Catostomus commersoni

Ictaluridae Channel catfish

Flathead catfish

Ictalurus punctatus

Pylodictis olivaris

Esocidae Northern pike Esox lucius

Salmonidae Lake trout

Brown trout

Rainbow trout

Salvelinus namaycush

Salmo trutta

Oncorhynchus mykissa

a

Formerly Salmo gairdneri.

Table 3-2. Recommended Target Species for Great Lakes Waters

Family name Common name Scientific name

Percichthyidae White bass Morone chrysops

Centrarchidae Smallmouth bass Micropterus dolomieui

Percidae Walleye Stizostedion vitreum

Cyprinidae Common carp Cyprinus carpio

Catostomidae White sucker Catostomus commersoni

Ictaluridae Channel catfish Ictalurus punctatus

Esocidae Muskellunge Esox masquinongy

Salmonidae Chinook salmon Oncorhynchus tschawytscha

Lake trout Salvelinus namaycush

Brown trout Salmo trutta

Rainbow trout Oncorhynchus mykissa

a

Formerly Salmo gairdneri.
3. TARGET SPECIES

3-5

extent of freshwater fish tissue contamination nationwide are compared in

Table 3-3. 

In addition to finfish species, states should consider monitoring the tissues of

freshwater turtles for environmental contaminants in areas where turtles are

consumed by recreational, subsistence, or ethnic populations. Interest has been

increasing in the potential transfer of environmental contaminants from the aquatic

food chain to humans via consumption of freshwater turtles. Turtles may

bioaccumulate environmental contaminants in their tissues from exposure to

contaminated sediments or via consumption of contaminated prey. Because

some turtle species are long-lived and occupy a medium to high trophic level of

the food chain, they have the potential to accumulate high concentrations of

chemical contaminants from their diets (Hebert et al., 1993). Some suggested

target turtle species for use in state contaminant monitoring programs are listed

in Table 3-4.

The list of target turtle species recommended for freshwater ecosystems was

developed based on a review of turtle species cited in state consumption

advisories or bans (RTI, 1993) and a review of the recent scientific literature. The

recommended target species list has been reviewed by regional and state

experts.

3.3.1 Target Finfish Species

3.3.1.1 Bottom-Feeding Species

EPA recommends that, whenever practical, states use common carp (Cyprinus

carpio), channel catfish (Ictalurus punctatus), and white sucker (Catostomus

commersoni) in that order as bottom-feeding target species in both inland fresh

waters (Table 3-1) and in Great Lakes waters (Table 3-2). These bottom-feeders

have been used consistently for monitoring a wide variety of contaminants

including dioxins/furans (Crawford and Luoma, 1993; U.S. EPA, 1992c, 1992d;

Versar Inc., 1984), organochlorine pesticides (Crawford and Luoma, 1993;

Schmitt et al., 1983, 1985, 1990; U.S. EPA, 1992c, 1992d), and heavy metals

(Crawford and Luoma, 1993; Lowe et al., 1985; May and McKinney, 1981;

Schmitt and Brumbaugh, 1990; U.S. EPA, 1992c, 1992d). These three species

are commonly consumed in the areas in which they occur and have also

demonstrated an ability to accumulate high concentrations of environmental

contaminants in their tissues as shown in Tables 3-5 and 3-6. Note: The

average contaminant concentrations shown in Tables 3-5 and 3-6 for fish

collected for the EPA National Study of Chemical Residues in Fish (U.S. EPA,

1992c, 1992d) were derived from concentrations in fish from undisturbed areas

and from areas expected to have elevated tissue contaminant concentrations.

The mean contaminant concentrations shown, therefore, may be higher or lower

than those found in the ambient environment because of site selection criteria

used in this study.
3. TARGET SPECIES

3-6

U.S. EPA

National

Dioxin Study

U.S. FWS

NPMP and NCBP

U.S. EPA

NSCRF

USGS

NWQAP

BOTTOM FEEDERS

Family Cyprinidae

Carp (Cyprinus carpio) ê ê êê

Family Icataluridae

Channel catfish (Ictalurus punctatus) ê

ê

Or other ictalurid ê ê

Family Catostomidae

White sucker (Catastomus commersoni)

ê ê

Or other catostomid

ê ê

Longnose sucker (C. catostromus) ê

Largescale sucker (C.macrocheilus)

Spotted sucker (Minytrema melanops) ê

Redhorse sucker (Moxostoma sp.)

included variety of species:

Silver redhorse (M. anisurum)

Grey redhorse (M. congetum)

Black redhorse (M. duquesnei)

Golden redhorse (M. erythrurum)

Shorthead redhorse (M. macrolepidotum)

Blacktail redhorse (M. poecilurum)

ê

PREDATORS

Family Salmoridae

Rainbow trout (Oncortynchus mykiss)

[formerly Salmo gairdneni]

Brown trout (Salmo trutta)

ê

ê

ê

ê êê

Brook trout (Salvelinus fontinalis) êê ê

Lake trout (Salmo namaycush) ê ê

Family Percidae

Walleye (Stizostedion vitreum) ê

Or other pericid

ê

Or other pericid

ê

Sauger (Stizostedion canadense)  

Yellow perch (Perca flavescans)  

Family Percichthyidae

White bass (Morone chrysops) ê

Family Centrarchidae

Largemouth bass (Micropterus salmoides) ê

Or other centrarchid

ê

Or other centrarchid

ê ê

Smallmouth bass (Micropterus dolomieui) ê

Black crappie (Pomoxis nigromaculatus)  

White crappie (Pomoxis annularis)  ê

Bluegill sunfish (Lepornis macrochirus)  ê

Family Esocidae

Northern pike (Esox lucius) ê

Family Ictaluridae

Flathead catfish (Pylodictis olivaris) ê

ê Recommended target species

 Alternate target species

NPMP = National Pesticide Monitoring Program

NCBP = National Contaminant Biomonitoring Program

NSCRF = National Study of Chemical Residues in Fish

NWQAP = National Water Quality Assessment Program

Sources: Versar, Inc., 1984; Schmitt et al., 1990; Schmitt et al., 1983; May and McKinney, 1981; U.S. EPA, 1992c, 1992d;

Crawford and Luoma, 1993.

Table 3-3. Comparison of Freshwater Finfish Species Used in Several National

Fish Contaminant Monitoring Programs
3. TARGET SPECIES

3-7

Table 3-4. Freshwater Turtles Recommended for Use as Target Species

Family name Common name Scientific name

Chelydridae Snapping turtle Chelydra serpentina

Emydidae Yellow-bellied turtle

Red-eared turtle

River cooter

Suwanee cooter

Slider

Texas slider

Florida cooter

Peninsula cooter

Trachemys scripta scripta

Trachemys scripta elegans

Pseudemys concinna concinna

Pseudemys concinna suwanniensis

Pseudemys concinna hieroglyphica

Pseudemys concinna texana

Pseudemys floridana floridana

Pseudemys floridana penisularis

Trionychidae Smooth softshell

Eastern spiny softshell

Western spiny softshell

Gulf Coast spiny softshell

Florida softshell

Apalone muticus

Apalone spinifera spinifera

Apalone spinifera hartwegi

Apalone spinifera aspera

Apalone ferox

In addition, these three species are relatively widely distributed throughout the

continental United States, and numerous states are already sampling these

species in their contaminant monitoring programs. A review of the database

National Listing of State Fish and Shellfish Consumption Advisories and Bans

(RTI, 1993) indicated that the largest number of states issuing advisories for

specific bottom-feeding species did so for carp (21 states) and channel catfish (22

states), with eight states issuing advisories for white suckers (see Table 3-7).

Appendix D lists the freshwater fish species cited in consumption advisories for

each state as of 1998.

3.3.1.2 Predator Species

EPA recommends that, whenever practical, states use predator target species

listed in Tables 3-1 and 3-2 for inland fresh waters and Great Lakes waters,

respectively. Predator species, because of their more definitive habitat and water

temperature preferences, generally have a more limited geographic distribution.

Thus, a greater number of predator species than bottom feeders have been used

in national contaminant monitoring programs (Table 3-3) and these are

recommended for use as target species in freshwater ecosystems. Predator fish

that prefer relatively cold freshwater habitats include many members of the

following families: Salmonidae (trout and salmon), Percidae (walleye and yellow

perch), and Esocidae (northern pike and muskellunge). Members of the

Centrarchidae (large- and smallmouth bass, crappie, and sunfish), Percichthyidae

(white bass), and Ictaluridae (flathead catfish) families prefer relatively warm

water habitats. Only two predator species (brown trout and largemouth bass)

were used in all four of the national monitoring programs reviewed by the 1993

EPA Fish Contaminant Workgroup (Table 3-3). However, most of the other

predator species recommended as target species have been used in at least one

national monitoring program. To identify those predator species with a known

ability to bioaccumulate contaminants in their tissues, the 1993 EPA Workgroup

reviewed average tissue concentrations of xenobiotic contaminants for major
3. TARGET SPECIES

3-8

Table 3-5. Average Fish Tissue Concentrations (ppb) of Xenobiotics for Major Finfish Species

Sampled in the National Study of Chemical Residues in Fisha

Fish Species Alpha- BHC Gamma- BHC Biphenyl Chlorpyrifos Dicofol Dieldrin Endrin Heptachlor epoxide Mercury Mirex Oxychlor- dane PCBs

Bottom Feedersb

Carp 3.10 4.34 4.38 8.23 0.88 44.75 1.40 4.00 0.11 3.70 8.20 2941.13White sucker 3.31 1.66 1.28 1.75 0.48 22.75 0.24 1.09 0.11 4.35 3.10 1697.81Channel cat 2.87 3.17 1.24 6.97 0.59 15.44 9.07 0.50 0.09 14.59 6.41 1300.52Redhorse sucker 0.82 0.41 1.25 0.35 ND 5.35 0.97 ND 0.27 0.57 2.37 487.72Spotted sucker 1.45 2.63 3.35 0.56 0.05 5.52 ND ND 0.12 1.79 0.05 133.90PredatorsbLargemouth bass 0.15 0.07 0.38 0.23 0.20 5.01 ND 0.30 0.46 0.21 0.47 232.26Smallmouth bass 0.36 0.15 0.33 0.08 ND 2.34 ND 0.07 0.34 1.99 0.54 496.22Walleye ND ND 0.40 0.04 ND 3.73 ND 0.21 0.51 0.08 1.11 368.65

Brown trout 1.59 ND 0.81 ND 0.94 20.13 ND 2.08 0.14 43.98 5.38 2434.07

White bass 0.34 0.79 0.62 1.32 ND 9.35 ND 1.40 0.35 0.11 0.84 288.35Northern pike 0.55 ND 0.59 11.43 0.31 9.04 ND ND 0.34 2.39 4.00 788.40Flathead cat 0.92 0.58 0.60 22.57 1.28 37.38 3.45 0.57 0.27 ND 0.63 521.19White crappie 0.23 ND 0.21 ND ND ND ND ND 0.22 ND ND 22.34

Bluefish 0.38 0.12 0.20 ND ND 2.87 ND ND 0.22 0.13 ND 368.06

Fish Species

Pentachloroanisole Penta- chloro- benzene DDE Total Chlordane Total Nonachlor 123 TCB 124 TCB 135 TCB 1234 TECB Trifluralin Hexa- chloro- benzene

Bottom Feedersb

Carp 16.50 1.04 415.43 67.15 63.15 1.54 4.77 0.08 0.30 12.55 3.58White sucker 9.06 0.39 78.39 18.43 20.83 0.16 0.30 0.14 0.15 ND 3.62Channel cat 39.60 1.32 627.77 54.39 66.28 0.14 0.37 ND 0.88 1.00 2.36Redhorse sucker 2.87 0.02 87.25 16.48 30.73 0.55 6.48 0.08 0.09 ND 0.58Spotted sucker 17.68 0.02 75.31 12.33 15.00 3.34 12.00 1.00 0.09 ND 0.02PredatorsbLargemouth bass 0.57 0.02 55.72 2.89 4.21 0.22 0.19 0.03 0.01 ND 0.20Smallmouth bass 0.23 0.02 33.63 4.01 7.82 0.70 0.59 0.04 0.04 ND 0.36Walleye 0.76 ND 34.00 3.62 8.04 0.29 0.38 ND 0.004 ND 0.11

Brown trout 0.09 0.60 158.90 7.25 32.60 1.10 0.98 ND 0.09 ND 3.06

White bass 0.93 ND 17.44 10.67 16.00 0.21 0.10 ND 0.01 ND 0.83Northern pike 1.51 0.09 59.50 5.45 13.88 0.30 0.23 ND 0.01 ND 0.20

Flathead catfish 0.31 ND 755.18 16.07 14.04 0.10 0.18 ND ND 44.37 0.85White crappie 0.33 ND 10.04 0.34 0.28 0.08 0.08 0.08 ND ND ND

Bluefish 0.05 ND 29.13 2.74 2.56 6.25 4.66 4.66 ND ND ND

a These average fish tissue concentrations may be higher or lower than those found in the ambient environment because of site selection criteria used in this study. b Values were calculated using whole-body samples for bottom feeders and fillet samples for predators. Individual values below detection limit were set at zero. Asterisk indicates all

values below the detection limit. Units = ppb, (μg/g) wet weight basis. ND = Not detected. 

Source: US. EPA, 1991h.
3. TARGET SPECIES

3-9

Table 3-6. Average Fish Tissue Concentrations (ppt) of Dioxins and Furans for Major Finfish Species

Sampled in the National Study of Chemical Residues in Fisha

Fish Species

2378

TCDD

12378

PeCDD

123478

HxCDD

123678

HxCDD

123789

HxCDD

1234678

HpCDD

2378

TCDF

12378

PeCDF

23478

PeCDF

123478

HxCDF

123678

HxCDF

123789

HxCDF

234678

HxCDF

1234678

HpCDF

1234789

HpCDF TEQ

Bottom Feedersb

Carp 7.76 3.63 2.16 6.81 1.54 22.29 10.15 1.31 4.01 2.54 1.91 1.16 1.20 2.49 1.22 13.06White sucker 8.08 2.05 1.03 1.96 0.88 3.72 22.89 1.10 2.64 2.21 1.29 1.06 1.09 1.23 1.13 12.79Channel catfish 11.56 2.37 1.61 5.62 1.29 9.40 2.22 0.52 2.91 2.41 1.41 1.38* 1.62 2.55 1.26 14.80Redhorse sucker 4.65 1.50 1.40 2.36 0.84 4.94 30.09 0.75 1.28 2.10 1.16 1.19* 1.50 1.57 1.36* 9.22Spotted sucker 1.73 2.34 1.70 12.08 1.14 17.48 7.49 2.12 2.06 2.22 1.79 1.28* 1.78 1.77 1.08 6.23PredatorsbLargemouth bass 1.73 0.59 1.12 1.28 0.64 2.48 2.18 0.37 0.47 1.24 1.23 1.21* 0.88 0.82 1.21* 1.91Smallmouth bass 0.72 0.50* 1.13* 0.79 0.64* 0.67 1.93 0.36* 0.51 1.28 1.23 1.26* 0.89* 0.69 1.30* 0.65*Walleye 0.88 0.54* 0.99* 0.73 0.62* 0.88 1.83 0.35* 0.38 1.04 1.09* 1.07* 0.75 0.74 1.21* 0.79*Brown trout 2.52 1.01 1.07* 0.98 0.68* 1.18 3.74 0.60 1.36 1.47 1.12* 1.09* 0.94* 0.67* 1.16* 3.31White bass 3.00 0.66 1.05* 0.78 0.61* 1.01 5.07 0.40 0.49 1.04 1.16* 1.13* 0.81* 0.63 1.17* 3.44Northern pike 0.77 0.46* 1.23* 0.91 0.69* 0.73 1.01 0.44 0.66 1.41* 1.42* 1.38* 0.98* 0.56 1.30* 0.66Flathead cat 0.78 0.43 0.90 1.06 0.50 1.67 1.63 0.40 0.56 1.05 1.20* 1.17* 0.61* 0.56 1.10* 0.99White crappie 2.13 0.60 1.29* 1.03* 0.83* 1.33 10.46 0.54 0.67 1.33* 1.33* 1.30* 0.95* 0.96* 1.34* 3.80Bluefish 0.85 0.56 1.23* 0.98* 0.69* 0.65 2.11 0.41 0.59 1.42* 1.42* 1.39* 0.98* 0.72* 1.31* 1.41

a These average fish tissue concentrations may be higher or lower than those found in the ambient environment because of site selection criteria used in this study.b Values were calculated using whole-body samples for bottom feeders and fillet samples for predators. Values below detection limit have been replaced by one-half detection limit for

the given sample. Asterisk indicates all values below detection limit. Units = ppt (pg/g) wet weight basis.TEQ = Toxicity equivalency was based on TEF-89 toxicity weighting values; however, octachlorodibenzo-p-dioxin and octachlorodibenzofurans were not analyzed; therefore, the TEQvalue does not include these two compounds.

Source: U.S. EPA, 1991h.
3. TARGET SPECIES

3-10

predator fish species sampled in the National Study of Chemical Residues in Fish.

Unlike the bottom feeders (common carp, channel catfish, and white suckers), no

single predator species or group of predator species consistently exhibited the

highest tissue concentrations for the contaminants analyzed (Tables 3-5 and 3-6).

However, average fish tissue concentrations for some contaminants (i.e.,

mercury, mirex, chlorpyrifos, DDE, 1,2,3-trichlorobenzene [123-TCB], and

trifluralin) were higher for some predator species than for the bottom feeders

despite the fact that only the fillet portion rather than the whole body was analyzed

for predator species. This finding emphasizes the need for using two types of fish

(i.e., bottom feeders and predators) with different habitat and feeding strategies

as target species.

The existence of fish consumption advisories for these predator target species

was further justification for their recommended use. As was shown for the

bottom-feeder target species, states were already sampling the recommended

predator target species listed in Table 3-7. The largest number of states issuing

advisories in 1993 for specific predator species did so for largemouth bass (15),

lake trout (10), white bass (10), smallmouth bass (9), brown trout (9), walleye (9),

rainbow trout (8), yellow perch (8), chinook salmon (7), northern pike (7), black

crappie (5), flathead catfish (4), and muskellunge (4) (RTI, 1993). For

comparison, the number of states reporting advisories for each species in 1998

is also presented in Table 3-7.

Because some freshwater finfish species (e.g., several Great Lake salmonids) are

highly migratory, harvesting of these species may be restricted to certain seasons

because sexually mature adult fish (i.e., the recommended size for sampling) may

make spawning runs from the Great Lakes into tributary streams. EPA recommends that spawning populations not be sampled in fish contaminant monitoring

programs. Sampling of target finfish species during their spawning period should

be avoided because contaminant tissue concentrations may decrease during this

time (Phillips, 1980) and because the spawning period is generally outside the

legal harvest period. Note: Target finfish may be sampled during their spawning

period, however, if the species can be legally harvested at this time.

State personnel, with their knowledge of site-specific fisheries and human

consumption patterns, must be the ultimate judge of the species selected for use

in freshwater fish contaminant monitoring programs within their jurisdiction.

3.3.2 Target Turtle Species

EPA recommends that states in which freshwater turtles are consumed by recreational, subsistence, or ethnic populations consider monitoring turtles to assess the

level of environmental contamination and whether they pose a human health risk.

In all cases, the primary criterion for selecting the target turtle species is whether

it is commonly consumed. To identify those turtle species with a known ability to

bioaccumulate contaminants in their tissues, the 1993 EPA Workgroup reviewed

turtle species cited in state consumption advisories and those species identified
3. TARGET SPECIES

3-11

Table 3-7. Principal Freshwater Fish Species Cited in State Fish

Consumption Advisoriesa

Family name Common name Scientific name

Number of states with advisoriesb

1993 1998

Percichthyidae White bass

Striped bass

White perch

Morone chrysops

Morone saxatilis

Morone americana

10

 6

 4

17

12

7

Centrarchidae Largemouth bass

Smallmouth bass

Black crappie

White crappie

Bluegill sunfish

Rock bass

Micropterus salmoides

Micropterus dolomieui 

Pomoxis nigromaculatus 

Pomoxis annularis

Lepomis macrochirus

Ambloplites rupestris

15

 9

 5

 2

 5

 3

33

18

18

11

11

5

Percidae Yellow perch

Sauger

Walleye

Perca flavescens

Stizostedion canadense

Stizostedion vitreum

8

 4

 9

12

9

12

Cyprinidae Common carp Cyprinus carpio 21 25

Acipenseridae Shovelnose sturgeon

Lake sturgeon 

Scaphirhynchus platorynchus

Acipenser fulvescens

 1

 2

3

3

Catostomidae Smallmouth buffalo

Bigmouth buffalo

Shorthead redhorse

White sucker

Quillback carpsucker

Ictiobus bubalus

Ictiobus cyprinellus

Moxostoma macrolepidotum

Catostomus commersoni 

Carpiodes cyprinus

 4

 4

 2

8

 2

5

6

3

11

5

Ictaluridae White catfish

Channel catfish

Flathead catfish

Black bullhead

Brown bullhead

Yellow bullhead

Ictalurus catus

Ictalurus punctatus

Pylodictis olivaris

Ictalurus melas

Ictalurus nebulosus

Ictalurus natalis

 5

22

 4

 2

 7

 2

6

26

11

3

10

8

Sciaenidae Freshwater drum Aplodinotus grunniens 313

Esocidae Northern pike

Muskellunge

Esox lucius 

Esox masquinongy

 7

 4

10

4

Salmonidae Coho salmon

Chinook salmon

Brown trout

Lake trout

Rainbow trout

Brook trout

Lake whitefish

Oncorhynchus kisutch

Oncorhynchus tschawytscha

Salmo trutta 

Salvelinus namaycush

Oncorhynchus mykissc

Salvelinus fontinalis

Coregonus clupea formis

 6

7

9

10

8

 3

 2

8

7

11

12

12

4

7

Anguillidae American eel Anguilla rostrata 6 7

a Species in boldface are EPA-recommended target species for inland fresh waters (see Table 3-1) and the Great

Lakes waters (Table 3-2). b Many states did not identify individual species of finfish in their advisories. c Formerly Salmo gairdneri.

Sources: RTI, 1993; U.S. EPA, 1999c (NLFWA).
3. TARGET SPECIES

3-12

Figure 3-1. Geographic range of the common snapping turtle 

(Chelydra serpentina).

in the scientific literature as having accumulated high concentrations of environmental contaminants. 

Based on information in state advisories and a number of environmental studies

using turtles as biological indicators of pollution, one species stands out as an

obvious choice for a target species, the common snapping turtle (Chelydra

serpentina). This turtle has been recommended by several researchers as an

important bioindicator species (Bishop et al., 1996; Bonin et al., 1995; Olafsson

et al., 1983; Stone et al., 1980) and has the widest geographic distribution of any

of the North American aquatic turtles (see Figure 3-1). In addition, this species

is highly edible, easily identified, easily collected, long-lived (>20 years), grows to

a large size, and has been extensively studied with respect to a variety of

environmental contaminants. Other turtle species that should be considered for

use as target species are listed in Table 3-4. 

Four states (Arizona, Massachusetts, Minnesota, and New York) currently have

consumption advisories in force for various turtle species (U.S. EPA, 1999c; New

York State Department of Health, 1994). The species cited in the state advisories

and the pollutants identified in turtle tissues as exceeding acceptable levels of

contamination with respect to human health are listed in Table 3-8. New York
3. TARGET SPECIES

3-13

Table 3-8. Principal Freshwater Turtle Species Cited in State Consumption Advisories

Family name Common name Scientific name Pollutant State

Chelydridae Snapping turtlea Chelydra serpentina Mercury MN

Snapping turtlea

(and other unspecified turtle

species)

Chelydra serpentina PCBs MA

Snapping turtleb Chelydra serpentina PCBs NY

Trionychidae Western spiny softshella Apalone spiniferus DDT

toxaphene,

chlordane,

dieldrin

AZ

PCB = Polychlorinated biphenyls. DDT = 1,1,1-trichloro-2,2 bis(p-chlorophenyl)ethane.

a

Source: U.S. EPA 1999c (NLFWA).

b

Source: New York State Department of Health, 1994.

state has a statewide advisory directed specifically at women of childbearing age

and children under 15 and advises these groups to avoid eating snapping turtles

altogether. The advisory also recommends that members of the general

population who wish to consume turtle meat should trim away all fat and discard

the liver tissue and eggs of the turtles prior to cooking the meat or preparing other

dishes. These three tissues (fat, liver, and eggs) have been shown to accumulate

extremely high concentrations of a variety of environmental contaminants in

comparison to muscle tissue (Bishop et al., 1996; Bonin et al., 1995; Bryan et al.,

1987; Hebert et al., 1993; Olafsson et al 1983; 1987; Ryan et al., 1986; Stone et

al., 1980). The Minnesota advisory also recommends that consumers remove all

fat from turtle meat prior to cooking as a risk-reducing strategy (Minnesota

Department of Health, 1994). States should consider monitoring pollutant

concentrations in all three tissues (fat, liver, and eggs) in addition to muscle tissue

if resources allow. If residue analysis reveals the presence of high concentrations

of any environmental contaminant of concern, the state should consider making

the general recommendation to consumers to discard these three highly lipophilic

tissues (fat, liver, and eggs) to reduce the risk of exposure particularly to many

organic chemical contaminants.

To identify those freshwater turtle species with a known ability to bioaccumulate

chemical contaminants in their tissues, several studies were reviewed that

identified freshwater turtle species as useful biomonitors of PCBs (Bishop et al.,

1996; Bonin et al., 1995; Bryan et al., 1987; Hebert et al., 1993; Helwig and Hora,

1983; Olafsson et al., 1983; 1987; Safe, 1985; and Stone et al., 1980), dioxins

and dibenzofurans (Bishop et al., 1996; Rappe et al., 1981; Ryan et al., 1986),

organochlorine pesticides (Bishop et al., 1996; Bonin et al., 1995; Hebert et al.,

1993; Stone et al., 1980), heavy metals (Bonin et al., 1995; Helwig and Hora,

1983; Stone et al., 1980), and radioactive nuclides (cesium-137 and strontium-90)

(Lamb et al., 1991; Scott et al., 1986). The turtle species used in these studies,

the pollutants monitored, and the reference sources are summarized in Table 3-9.
3. TARGET SPECIES

3-14

Table 3-9. Studies Using Freshwater Turtles as Biomonitors of

Environmental Contamination

Species Pollutant monitored Source

Snapping turtle

(Chelydra serpentina)

PCBs, total DDT, mirex Hebert et al., 1993

Snapping turtle

(Chelydra serpentina)

PCBs Olafsson et al., 1987

Olafsson et al., 1983

Snapping turtle

(Chelydra serpentina)

PCBs Safe, 1987

Snapping turtle

(Chelydra serpentina)

PCBs Bryan et al., 1987

Snapping turtle

(Chelydra serpentina)

Dioxins/Furans Ryan et al., 1986

Snapping turtle

(Chelydra serpentina)

PCBs, mercury, cadmium Helwig and Hora, 1983

Snapping turtle

(Chelydra serpentina)

Furans Rappe et al., 1981

Snapping turtle

(Chelydra serpentina)

Organochlorine pesticides

(DDE, dieldrin, hexachlorobenzene,

heptachlor epoxide, mirex), PCBs,

cadmium, mercury

Stone et al., 1980

Snapping turtle

(Chelydra serpentina) 29 Organochlorine pesticides,

39 PCB congeners, mercury

Bonin et al., 1995

Snapping turtle eggs 4 Organochlorine pesticides

(DDE, dieldrin, mirex, hexachlorobenzene), PCBs, dioxins/furans

Bishop et al., 1996

Yellow-bellied turtle

(Trachemys scripta)

Cesium-137

Strontium-90

Lamb et al., 1991

Yellow-bellied turtle

(Trachemys scripta)

Cesium-137

Strontium-90

Scott et al., 1986

PCBs = Polychlorinated biphenyls. 

DDT = 1,1,1-Trichloro-2,2 bis(p-chlorophenyl)ethane.

DDE = 1,1-Dichloro-2,2-bis(p-chlorophenyl)-ethylene.

State personnel, with their knowledge of site-specific fisheries and human

consumption patterns, must be the ultimate judge of the turtle species selected

for use in contaminant monitoring programs within their jurisdictions. Because

several turtle species are becoming less common as a result of habitat loss or

degradation or overharvesting, biologists need to ensure that the target species

selected for the state toxics monitoring program is not of special concern within

their jurisdiction or designated as a threatened or endangered species. For

example, two highly edible turtle species, the Alligator snapping turtle

(Macroclemys temmincki) and the Northern diamondback terrapin (Malaclemys

terrapin terrapin) are protected in some states or designated as species of

concern within portions of their geographic range and are also potential

candidates for federal protection (Sloan and Lovich, 1995). Although protected

to varying degrees by several states, George (1987) and Pritchard (1989)

concluded that the Alligator snapping turtle should receive range-wide protection
3. TARGET SPECIES

3-15

from the federal government as a threatened species under the Endangered

Species Act. Unfortunately, basic ecological and life history information

necessary to make environmental management decisions (i.e., federal listing as

endangered or threatened species) is often not available for turtles and other

reptiles (Gibbons, 1988). 

Several species of freshwater turtles already have been designated as

endangered or threatened species in the United States including the Bog turtle

(Clemmys muhlenbergii), Plymouth red-bellied turtle (Pseudemys rubriventris

bangsi), Alabama red-bellied turtle (Pseudemys alabamensis), Flattened musk

turtle (Stemotherus depressus), Ringed map (=sawback) turtle (Graptemys

oculifera), and the Yellow-blotched map (=sawback) turtle (Graptemys

flavimaculata) (U.S. EPA, 1994; U.S. Fish and Wildlife Service, 1994). In addition,

all species of marine sea turtles including the Green sea turtle (Chelonia mydas),

Hawksbill sea turtle (Eretmochelys imbricata), Kemp’s ridley sea turtle

(Lepidochelys kempii), Olive ridley sea turtle (Lepidochelys olivacea), Loggerhead

sea turtle (Caretta caretta), and the Leatherback sea turtle (Dermochelys

coriacea) have been designated as endangered (U.S. EPA, 1994; U.S. Fish and

Wildlife Service, 1994). 

3.4 ESTUARINE/MARINE TARGET SPECIES

EPA recommends that states collect either one shellfish species (preferably a

bivalve mollusc) and one finfish species or two finfish species at each

estuarine/marine screening site. In all cases, the primary criterion for selecting

the target species is that it is commonly consumed. Ideally, one shellfish species

and one finfish species should be sampled; however, if no shellfish species from

the recommended target species list meets the primary criterion, EPA

recommends that states use two finfish species selected from the appropriate

regional estuarine/marine target species lists. If two finfish are selected as the

target species, one should be a bottom-feeding species.

EPA recommends that, whenever practical, states use target species selected

from fish and shellfish species identified in Tables 3-10 through 3-16 for the

following specific estuarine/marine coastal areas:

• Northeast Atlantic region (Maine through Connecticut)—Table 3-10

• Mid-Atlantic region (New York through Virginia)—Table 3-11

• Southeast Atlantic region (North Carolina through Florida)—Table 3-12

• Gulf Coast region (west coast of Florida through Texas)—Table 3-13

• Pacific Northwest region (Alaska through Oregon)—Table 3-14

• Northern California waters (Klamath River through Morro Bay)—Table 3-15

• Southern California waters (Santa Monica Bay to Tijuana Estuary)—

Table 3-16.
3. TARGET SPECIES

3-16

Table 3-10. Recommended Target Species for Northeast Atlantic

Estuaries and Marine Waters (Maine through Connecticut)

Family name Common name Scientific name

Finfish Species

Anguillidae American eel Anguilla rostrata

Percichthyidae Striped bass Morone saxatilis

Pomatomidae Bluefish Pomatomus saltatrix

Sparidae Scup Stenotomus chrysops

Sciaenidae Weakfish Cynoscion regalis

Bothidae Summer flounder Paralichthys dentatus

Four-spotted flounder Paralichthys oblongus

Pleuronectidae Winter flounder Pseudopleuronectes

americanus

Yellowtail flounder Limanda ferruginea

American dab Hippoglossoides

platessoides

Shellfish Species

Bivalves Soft-shell clam Mya arenaria

Mercenaria mercenaria

Arctica islandica

Spisula solidissima

Mytilus edulis

Crustaceans American lobster

Eastern rock crab

Homarus americanus

Cancer irroratus
3. TARGET SPECIES

3-17

Table 3-11. Recommended Target Species for Mid-Atlantic

Estuaries and Marine Waters (New York through Virginia)

Family name Common name Scientific name

Finfish Species

Anguillidae American eel Anguilla rostrata

Ictaluridae Channel catfish Ictalurus punctatus

White catfish Ictalurus catus

Percichthyidae White perch Morone americana

Striped bass Morone saxatilis

Pomatomidae Bluefish Pomatomus saltatrix

Sparidae Scup Stenotomus chrysops

Sciaenidae Weakfish Cynoscion regalis

Spot Leistomus xanthurus

Atlantic croaker Micropogonias undulatus

Red drum Sciaenops ocellatus

Bothidae Summer flounder Paralichthys dentatus

Pleuronectidae Winter flounder Pseudopleuronectes americanus

Shellfish Species

Bivalves Hard clam Mercenaria mercenaria

Soft-shell clam Mya arenaria

Ocean quahog Arctica islandica

Surf clam Spisula solidissima

Blue mussel Mytilus edulis

American oyster Crassostrea virginica

Crustaceans Blue crab Callinectes sapidus

American lobster Homarus americanus

Eastern rock crab Cancer irroratus
3. TARGET SPECIES

3-18

Table 3-12. Recommended Target Species for Southeast Atlantic

Estuaries and Marine Waters (North Carolina through Florida)

Family name Common name Scientific name

Finfish Species

Anguillidae American eel Anguilla rostrata

Ictaluridae Channel catfish

White catfish

Ictalurus punctatus

Ictalurus catus

Percichthyidae White perch

Striped bass

Morone americana

Morone saxatilis

Sciaenidae Spot Leistomus xanthurus

Atlantic croaker Micropogonias undulatus

Red drum Sciaenops ocellatus

Bothidae Southern flounder Paralichthys lethostigma

Summer flounder Paralichthys dentatus

Shellfish Species

Bivalves Hard clam Mercenaria mercenaria

American oyster Crassostrea virginica

Crustaceans West Indies spiny lobster Panulirus argus

Blue crab Callinectes sapidus
3. TARGET SPECIES

3-19

Table 3-13. Recommended Target Species for Gulf of Mexico

Estuaries and Marine Waters (West Coast of Florida through Texas)

Family name Common name Scientific name

Finfish Species

Ictaluridae Blue catfish Ictalurus furcatus

Channel catfish Ictalurus punctatus

Ariidae Hardhead catfish Arius felis

Sciaenidae Spotted seatrout Cynoscion nebulosus

Spot Leistomus xanthurus

Atlantic croaker Micropogonias undulatus

Red drum Sciaenops ocellatus

Bothidae Gulf flounder Paralichthys albigutta

Southern flounder Paralichthys lethostigma

Shellfish Species

Bivalves American oyster Crassostrea virginica

Hard clam Mercenaria mercenaria

Crustaceans White shrimp Penaeus setiferus

Blue crab Callinectes sapidus

Gulf stone crab Menippe adina

West Indies spiny lobster Panulirus argus
3. TARGET SPECIES

3-20

Table 3-14. Recommended Target Species for Pacific Northwest

Estuaries and Marine Waters (Alaska through Oregon)

Family name Common name Scientific name

Finfish Species

Embiotocidae Redtail Surfperch Amphistichus rhodoterus

Scorpaenidae Copper rockfish Sebastes caurinus

Black rockfish Sebastes melanops

Bothidae Speckled sanddab Citharichthys stigmaeus

Pacific sanddab Citharichthys sordidus

Pleuronectidae Starry flounder Platichthys stellatus

English sole Parophrys vetulus

Salmonidae Coho salmon Onchorhynchus kisutch

Chinook salmon Onchorhynchus tshawytscha

Shellfish Species

Bivalves Blue mussel Mytilus edulis

California mussel Mytilus californianus

Pacific oyster Crassostrea gigas

Horseneck clam Tresus capax

Pacific littleneck clam Protothaca staminea

Soft-shell clam Mya arenaria

Manila clam Venerupis japonica

Crustaceans Dungeness crab Cancer magister

Red crab Cancer productus
3. TARGET SPECIES

3-21

Table 3-15. Recommended Target Species for Northern California 

Estuaries and Marine Waters (Klamath River through Morro Bay)

Family name Common name Scientific name

Finfish Species

Triakidae Leopard shark Triakis semifasciata

Sciaenidae White croaker Genyonemus lineatus

Embiotocidae Redtailed surfperch Amphistichus rhodoterus

Striped seaperch Embiotoca lateralis

Scorpaenidae Black rockfish Sebastes melanops

Yellowtail rockfish Sebastes flavidus

Bocaccio Sebastes paucispinis

Bothidae Pacific sanddab Citharichthys sordidus

Speckled sanddab Citharichthys stigmaeus

Pleuronectidae Starry flounder Platichthys stellatus

English sole Parophrys vetulus

Salmonidae Coho salmon Onchorhynchus kisutch

Chinook salmon Onchorhynchus tshawytscha

Shellfish Species

Bivalves Blue mussel Mytilus edulis

California mussel Mytilus californianus

Pacific littleneck clam Protothaca staminea

Soft-shell clam Mya arenaria

Crustaceans Dungeness crab Cancer magister

Red crab Cancer productus

Pacific rock crab Cancer antennarius
3. TARGET SPECIES

3-22

Table 3-16. Recommended Target Species for Southern California

Estuaries and Marine Waters (Santa Monica Bay to Tijuana Estuary)

Family name Common name Scientific name

Finfish Species

Serranidae Kelp bass Paralabrax clathratus

Barred sand bass Paralabrax nebulifer

Sciaenidae White croaker Genyonemus lineatus

Corbina Menticirrhus undulatus

Embiotocidae Black perch Embiotoca jacksoni

Walleye surf perch Hyperprosopan argenteum

Barred surfperch Amphistichus argenteus

Scorpaenidae California scorpionfish Scorpaena guttata

Widow rockfish Sebastes entomelas

Blue rockfish Sebastes mystinus

Bocaccio Sebastes paucispinis

Pleuronectidae Diamond turbot Hypsopetta guttulata

Dover sole Microstomus pacificus

Shellfish Species

Bivalves Blue mussel Mytilus edulis

California mussel Mytilus californianus

Pacific littleneck clam Protothaca staminea

Crustaceans Pacific rock crab Cancer antennarius

Red crab Cancer productus

California rock lobster Panulirus interruptus

The seven separate regional lists of target species recommended by the 1993

EPA Workgroup for estuarine/marine ecosystems were developed because of

differences in species’ geographic distribution and abundance and the nature of

the regional fisheries and were developed based on a review of species used in

the following national monitoring programs:

• National Dioxin Study (U.S. EPA)

• Section 301(h) Monitoring Program (U.S. EPA)

• National Status and Trends Program (NOAA)

• National Study of Chemical Residues in Fish (U.S. EPA).

Because some of these programs identified some fish and shellfish species that

are not of commercial, sportfishing, or subsistence value, several additional

literature sources identifying commercial and sportfishing species were also
3. TARGET SPECIES

3-23

reviewed (Table 3-17). Some sources included information on seasonal

distribution and abundance of various life stages (i.e., adults, spawning adults,

juveniles) of fish and shellfish species. This information was useful in delineating

seven regional estuarine/marine areas nationwide. The 1993 EPA Workgroup

also reviewed fish and shellfish species cited in state consumption advisories for

estuarine/marine waters (Appendix D). Each of the final regional lists of target

species has been reviewed by state, regional, and national fisheries experts.

Use of two distinct ecological groups of organisms (shellfish and finfish) as target

species in estuarine/marine systems is recommended. This permits monitoring

of a wide variety of habitats, feeding strategies, and physiological factors that

might result in differences in bioaccumulation of contaminants. Estuarine/marine

species used in several national contaminant monitoring programs reviewed by

the 1993 EPA Workgroup are compared in Table 3-18.

3.4.1 Target Shellfish Species

Selection of shellfish species (particularly bivalve molluscs) as target species

received primary consideration by the 1993 EPA Workgroup because of the

commercial, recreational, and subsistence value of shellfish in many coastal areas

of the United States. Bivalve molluscs (e.g., oysters, mussels, and clams) are

filter feeders that accumulate contaminants directly from the water column or via

ingestion of contaminants adsorbed to phytoplankton, detritus, and sediment

particles. Bivalves are good bioaccumulators of heavy metals (Cunningham,

1979) and polycyclic aromatic hydrocarbons (PAHs) and other organic

compounds (Phillips, 1980; NOAA, 1987) and, because they are sessile, they may

reflect local contaminant concentrations more accurately than more mobile

crustacean or finfish species.

Three bivalve species—the blue mussel (Mytilus edulis), the California mussel

(Mytilus californianus), and the American oyster (Crassostrea virginica)—were

recommended and/or used in three of the national monitoring programs reviewed

by the 1993 EPA Workgroup. Two other bivalve species—the soft-shell clam

(Mya arenaria) and the Pacific oyster (Crassostrea gigas)—were also

recommended and/or used in two national programs. Although no bivalve species

was identified by name in state fish and shellfish consumption advisories

(Appendix D), seven coastal states issued advisories in 1993 for unspecified

bivalves or shellfish species that may have included these and other bivalve

species. All three species are known to bioaccumulate a variety of environmental

contaminants (Phillips, 1988). The wide distribution of these three species makes

them useful for comparison within a state or between states sharing coastal

waters (Figure 3-2). Because these three species met all of the selection criteria,

they were recommended as target species for use in geographic areas in which

they occur.
3. TARGET SPECIES

3-24

Table 3-17. Sources of Information on Commercial and Sportfishing

Species in Various Coastal Areas of the United States

Geographic

area Source

Atlantic Coast National Marine Fisheries Service. 1987. Marine Recreational Fishery Statistics Survey, Atlantic and Gulf

Coasts, 1986. Current Fishery Statistics Number 8392. National Oceanic and Atmospheric Administration,

U.S. Department of Commerce, Rockville, MD.

Leonard, D.L., M.A. Broutman, and K.E. Harkness. 1989. The Quality of Shellfish Growing Waters on the

East Coast of the United States. Strategic Assessment Branch, National Oceanic and Atmospheric

Administration, U.S. Department of Commerce, Rockville, MD.

Nelson, D.M., M.E. Monaco, E.A. Irlandi, L.R. Settle, and L. Coston-Clements. 1991. Distribution and

Abundance of Fishes and Invertebrates in Southeast Estuaries. ELMR Report No. 9. Strategic Assessment

Division. National Oceanic and Atmospheric Administration, U.S. Department of Commerce, Rockville, MD.

Stone, S.L., T.A. Lowery, J.D. Field, C.D. Williams, D.M. Nelson, S.H. Jury, M.E. Monaco, and L. Andreasen. 

1994. Distribution and Abundance of Fishes and Invertebrates in Mid-Altantic Estuaries. ELMR Rep. No. 12. 

NOAA/NOS Strategic Environmental Assessments Division, SIlver Spring, MD.

Jury, S.H., J.D. Field, S.L. Stone, D.M. Nelson, and M.E. Monaco. 1994. Distribution and Abundance of

Fishes and Invertebrates in North Atlantic Estuaries. ELMR Rep. No. 13. NOAA/NOS Strategic

Environmental Assessments Division, SIlver Spring, MD.

Gulf Coast National Marine Fisheries Service. 1987. Marine Recreational Fishery Statistics Survey, Atlantic and Gulf

Coasts, 1986. Current Fishery Statistics Number 8392. National Oceanic and Atmospheric Administration,

U.S. Department of Commerce, Rockville, MD.

Broutman, M.A., and D.L. Leonard. 1988. The Quality of Shellfish Growing Waters in the Gulf of Mexico.

Strategic Assessment Branch, National Oceanic and Atmospheric Administration, Rockville, MD.

Monaco, M.E., D.M. Nelson, T.C. Czapla, and M.E. Patillo. 1989. Distribution and Abundance of Fishes and

Invertebrates in Texas Estuaries. ELMR Report No. 3. Strategic Assessment Branch, National Oceanic and

Atmospheric Administration, U.S. Department of Commerce, Rockville, MD.

Williams, C.D., D.M. Nelson, M.E. Monaco, S.L. Stone, C. Iancu, L. Coston-Clements, L.R. Settle, and E.A.

Irlandi. 1990. Distribution and Abundance of Fishes and Invertebrates in Eastern Gulf of Mexico Estuaries.

ELMR Report No. 6. Strategic Assessment Branch, National Oceanic and Atmospheric Administration, U.S.

Department of Commerce, Rockville, MD.

Czapla, T.C., M.E. Patillo, D.M. Nelson, and M.E. Monaco. 1991. Distribution and Abundance of Fishes and

Invertebrates in Central Gulf of Mexico Estuaries. ELMR Report No. 7. Strategic Assessment Branch,

National Oceanic and Atmospheric Administration, U.S. Department of Commerce, Rockville, MD.

Nelson, D.M. (editor). 1992. Distribution and Abundance of Fishes and Invertebrates in Gulf of Mexico

Estuaries, Volume I: Data Summaries. ELMR Rep. No. 10. NOAA/NOS Strategic Environmental

Assessments Division, Rockville, MD.

Patillo, M.E., T.E. Czapla, D.M. Nelson, and M.E. Monaco. 1997. Distribution and Abundance of Fishes and

Invertebrates in Gulf of Mexico Estuaries. Vol. II: Species Life History Summaries . ELMR Rep. No. 14.

NOAA/NOS Strategic Environmental Assessments Division, Silver Spring, MD.

West Coast National Marine Fisheries Service. 1987. Marine Recreational Fishery Statistics Survey, Pacific Coast, 1986.

Current Fishery Statistics Number 8393. National Oceanic and Atmospheric Administration, U.S. Department

of Commerce, Rockville, MD.

Leonard, D.L., and E.A. Slaughter. 1990. The Quality of Shellfish Growing Waters on the West Coast of the

United States. Strategic Assessment Branch, National Oceanic and Atmospheric Administration, U.S.

Department of Commerce, Rockville, MD.

Monaco, M.E., D.M. Nelson, R.L. Emmett, and S.A. Hinton. 1990. Distribution and Abundance of Fishes and

Invertebrates in West Coast Estuaries. Volume I: Data Summaries. ELMR Report No. 4. Strategic

Assessment Branch, National Oceanic and Atmospheric Administration, Rockville, MD.

Emmett, R.L., S.A. Hinton, S.L. Stone, and M.E. Monaco. 1991. Distribution and Abundance of Fishes and

Invertebrates in West Coast Estuaries. Volume II: Life History Summaries. ELMR Report No. 8. Strategic

Environmental Assessment Division, Rockville, MD.
3. TARGET SPECIES

3-25

U.S. EPA

National

Dioxin Studya

NOAA

Status and

Trends

U.S. EPA

301(h)

Program

U.S. EPA

NSCRFb

FINFISH

Family Acipenseridae

White sturgeon (Acipenser transmontanus) ê

Family Ariidae

Hardhead catfish (Arius felis) ê

Family Percichthyidae

White perch (Morone americana) ê

Family Pomatomidae

Bluefish (Pomatomus saltatrix) ê

Family Lutjanidae

Red snapper (Lutjanus campechanus) ê

Family Sparidae

Sheepshead (Archosargus probatocephalus) ê

Family (Sciaenidae)

Spotted seatrout (Cynoscion nebulosus)

Weakfish (Cynoscion regalis)

Spot (Leiostomus xanthurus)

White croaker (Genyonemus lineatus)

Atlantic craoker (Micropogonias undulatus)

Black drum (Pogonias cromis)

Red drum (Sciaenops ocellatus)

ê

ê

ê

ê

ê

ê

ê

ê

ê

ê

Family Serranidae

Barred sand bass (Paralabrax nebulifer) ê

Family Mugilidae

Striped mullet (Mugil cephalus) ê

Family Bothidae

Southern flounder (Paralichthys lethostigma) ê

Windowpane flounder (Scophthalmus aquosus) ê

Family Pleuronectidae

Pacific sanddab (Citharichthys sordidus)

Flathead sole (Hippoglossoides elassodon)

Diamond turbot (Hypsopsetta guttulata)

Starry flounder (Platichthys stellatus)

Hornyhead turbot (Pleuronichthys verticalis)

Winter flounder (Pseudopleuronectes americanus)

English sole (Parophrys vetulus)

Dover sole (Microstomus pacificus)

ê

ê

ê

ê

ê

ê

ê

ê

ê

ê

ê

ê

ê

See notes at end of table. (continued)

Table 3-18. Estuarine/Marine Species Used in Several National Fish and Shellfish

Contaminant Monitoring Programs
3. TARGET SPECIES

3-26

Table 3-18. (continued)

U.S. EPA

National

Dioxin Studya

NOAA

Status and

Trends

U.S. EPA

301(h)

Program

U.S. EPA

NSCRFb

SHELLFISH

Bivalves

Hard clam (Mercenaria mercanaria)

Soft-shell clam (Mya arenaria)

Ocean quahog (Arctica islandia)

Surf clam (Spisula solidissima)

Blue mussel (Mytilus edulis)

California mussel (Mytilus californianus)

American oyster (Crassostrea virginica)

Hawaiian oyster (Ostrea sandwichensis)

Pacific oyster (Crassostrea gigas)

Bent-nosed macoma (Macoma nasuta)

Baltic macoma (Macoma baltica)

White sand macoma (Macoma secta)

ê

ê

ê

ê

ê

ê

ê

ê

ê

ê

ê

ê

ê

ê

ê

ê

ê

ê

ê

ê

Crustaceans

American lobster (Homarus americanus)

West Indies spiny lobster (Panulirus argus)

California rock lobster (Panulirus interruptus)

Hawaiian spiny lobster (Panulirus penicillatus)

Eastern rock crab (Cancer irroratus)

Dungeness crab (Cancer magister)

Pacific rock crab (Cancer antennarius)

Yellow crab (Cancer anthonyi)

Red crab (Cancer productus)

ê

ê

ê

ê

ê

ê

ê

ê

ê

ê

NSCRF = National Study of Chemical Residues in Fish.

a Only freshwater finfish were identified as target species; bivalves were identified as estuarine/marine target species. b Species listed were those collected at more than one site nationally; Salmonidae were not listed because they were included on

freshwater lists.
3. TARGET SPECIES

3-27

Source: Abbott, 1974.

Figure 3-2. Geographic distributions of three bivalve species used extensively in national contaminant

monitoring programs.
3. TARGET SPECIES

3-28

In addition, several species of edible clams were added to the various estuarine/

marine target species lists based on recommendations received from specific

state and regional fisheries experts.

Crustaceans are also recommended as target species for estuarine/marine

sampling sites. Many crustaceans are bottom-dwelling and bottom-feeding

predator and/or scavenger species that are good indicators of contaminants that

may be biomagnified through several trophic levels of the food web. Several

species of lobsters and crabs were recommended in one national monitoring

program, and the Dungeness crab was recommended in two national monitoring

programs (Table 3-18). These crustaceans, although of fishery value in many

areas, are not as widely distributed nationally as the three bivalve species (Figure

3-2). However, they should be considered for selection as target species in states

where they are commonly consumed.

Only two crustaceans—the American lobster (Homarus americanus) and the blue

crab (Callinectes sapidus)—were specifically identified in state advisories (RTI,

1993). However, in 1993, seven coastal states reported advisories in estuarine/

marine waters for unspecified shellfish species that may have included these and

other crustacean species (Table 3-19). All of the shellfish species cited in state

advisories are included as EPA-recommended target species on the appropriate

estuarine/marine regional lists.

3.4.2 Target Finfish Species

Two problems were encountered in the selection of target finfish species for

monitoring fish tissue contamination at estuarine/marine sites regionally and

nationally. First is the lack of finfish species common to both Atlantic and Gulf

Coast waters as well as Pacific Coast waters. Species used in several federal

fish contaminant monitoring programs are compared in Table 3-18. Members of

the families Sciaenidae (seven species), Bothidae (two species), and

Pleuronectidae (eight species) were used extensively in these programs. Bottomdwelling finfish species (e.g., flounders in the families Bothidae and

Pleuronectidae) may accumulate high concentrations of contaminants from direct

physical contact with contaminated bottom sediments. In addition, these finfish

feed on sedentary infaunal or epifaunal organisms and are at additional risk of

accumulating contaminants via ingestion of these contaminated prey species

(U.S. EPA, 1987a). For finfish species, two Atlantic coast species, spot

(Leiostomus xanthurus) and winter flounder (Pseudopleuronectes americanus),

are recommended and/or used in three of the national monitoring programs, and

the Atlantic croaker (Micropogonias undulatus) is recommended and/or used in

two national monitoring programs. Three Pacific coast species, Starry flounder

(Platichthys stellatus), English sole (Parophrys vetulus), and Dover sole

(Microstomus pacificus), are recommended or used in two of the national

monitoring programs.
3. TARGET SPECIES

3-29

Table 3-19. Principal Estuarine/Marine Fish and Shellfish Species Cited in State

Consumption Advisoriesa,b

Species group name Common name Scientific name

Number of

states with

advisories

in 1993

Number of

states with

advisories

in 1998

Finfish

Percichthyidae Striped bass

White perch

Morone saxatilis

Morone americana

5

3

6

3

 Centrarchidae Largemouth bass

Smallmouth bass

Micropterus salmoides

Micropterus dolomieui

0

0

3

1

Ictaluridae White catfish

Channel catfish

Ictalurus catus

Ictalurus punctatus

4

5

2

2

Anguillidae American eel Anguilla rostrata 6 5

 Elopidae Ladyfish Elops saurus 0 1

 Carangidae Crevalle jack Caranx hippos 0 1

Pomatomidae Bluefish Pomatomus saltatrix 4 6

 Labridae Tautog Tautoga onitis 0 1

 Sparidae Scup Stenotomus chrysops 0 1

 Sciaenidae Spotted sea trout

Atlantic croaker

Red drum

Black drum

Silver perch

Cynoscion nebulosus

Micropogonias undulatus

Sciaenops ocellatus

Pogonias cromis

Bairdiella chrysoura

0

0

0

0

0

2

1

1

1

1

 Scombridae King mackerel

Spanish mackerel

Scomberomorus cavalla

Scomberomorus maculatus

0

0

5

1

 Ariidae Gafftopsail catfish Bagre marinus 0 1

Belonidae Atlantic needlefish Strongylura marina 1 1

Serranidae Kelp bass Paralabrax clathratus 1 1

Sciaenidae Black croaker

White croaker

Queenfish

Corbina

Cheilotrema saturnum

Genyonemus lineatus

Seriphus politus

Menticirrhus undulatus

1

1

1

1

1

1

1

1

Shellfish

Crustaceansc American lobster

Blue crab

Homarus americanus

Callinectes sapidus

1

3

5

4

a Species in boldface are EPA-recommended target species for regional estuarine/marine waters (see Tables 3-10

through 3-16). b Many coastal states issued advisories for fish and shellfish species and thus did not identify specific finfish and

shellfish species in their advisories. c Eight coastal states (California, Georgia, Hawaii, Louisiana, Massachusetts, North Carolina, Texas, and

Washington) and the U.S. territory of American Samoa report advisories for unspecified shellfish or bivalve

species.

Sources: RTI, 1993, EPA 1999a (NLFWA).
3. TARGET SPECIES

3-30

Second, because some estuarine/marine finfish species are highly migratory,

harvesting of these species may be restricted to certain seasons because

sexually mature adult fish (i.e., the recommended size for sampling) may enter

the estuaries only to spawn. EPA recommends that neither spawning populations

nor undersized juvenile stages be sampled in fish contaminant monitoring

programs. Sampling of target finfish species during their spawning period should

be avoided as contaminant tissue concentrations may decrease during this time

(Phillips, 1980) and because the spawning period is generally outside the legal

harvest period. Note: Target finfish species may be sampled during their

spawning period if the species can be legally harvested at this time. Sampling of

undersized juveniles of species that use estuaries as nursery areas is precluded

by EPA’s recommended monitoring strategy because juveniles may not have had

sufficient time to bioaccumulate contaminants or attain harvestable size.

Because of these problems, the 1993 EPA Workgroup consulted with regional

and state fisheries experts and reviewed the list of state fish consumption

advisories and bans to determine which estuarine/marine finfish species should

be recommended as target species. As shown in Table 3-19, the largest number

of states issuing advisories in 1993 for specific estuarine and marine waters did

so for the American eel (6), channel catfish (5), striped bass (5), bluefish (4),

white catfish (4), and white perch (3). Several other estuarine/marine species

were cited in advisories for one state each (Table 3-19). Many coastal states did

not identify individual finfish species by name in their advisories (see Appendix D);

however, almost all of the species that have been cited in state advisories are

recommended as target species by EPA (see Tables 3-10 through 3-16). The

listing of estuarine fish and shellfish cited in state advisories in 1998 is also shown

in Table 3-19.

These seven regional lists of recommended estuarine/marine target species are

provided to give guidance to states on species commonly consumed by the

general population. state personnel, with their knowledge of site-specific fisheries

and human consumption patterns, must be the ultimate judge of the species

selected for use in estuarine/marine fish contaminant monitoring programs within

their jurisdiction.
4. TARGET ANALYTES

4-1

SECTION 4

TARGET ANALYTES

The selection of appropriate target analytes in fish and shellfish contaminant

monitoring programs is essential to the adequate protection of the health of fish

and shellfish consumers. The procedures used for selecting target analytes for

screening studies and a list of recommended target analytes are presented in this

section.

4.1 RECOMMENDED TARGET ANALYTES

Recommended target analytes for screening studies in fish and shellfish

contaminant monitoring programs are listed in Table 4-1. This list was developed

by the EPA 1993 Fish Contaminant Workgroup from a review of the following

information:

1. Pollutants analyzed in several national or regional fish contaminant

monitoring programs—The monitoring programs reviewed included

• National Study of Chemical Residues in Fish (U.S. EPA)

• National Dioxin Study (U.S. EPA)

• 301(h) Monitoring Program (U.S. EPA)

• National Pollutant Discharge Elimination System (U.S. EPA)

• National Pesticide Monitoring Program (U.S. FWS)

• National Contaminant Biomonitoring Program (U.S. FWS)

• National Status and Trends Program (NOAA)

• Great Lakes Sportfish Consumption Advisory Program

• National Water Quality Assessment Program (USGS).

Criteria for selection of the target analytes in these programs varied widely

depending on specific program objectives. The target analytes used in these

major fish contaminant monitoring programs are compared in Appendix E.

Over 200 potential contaminants are listed, including metals, pesticides,

base/neutral organic compounds, dioxins, dibenzofurans, acidic organic

compounds, and volatile organic compounds.
4. TARGET ANALYTES

4-2

Table 4-1. Recommended Target Analytes

Metals

Arsenic (inorganic)

Cadmium 

Mercury (methylmercury)

Selenium

Tributyltin

Organochlorine Pesticides

Chlordane, total (cis- and trans-chlordane,

cis- and trans-nonachlor, oxychlordane)

DDT, total (2,4’-DDD, 4,4’-DDD, 2,4’-DDE,

4,4’-DDE, 2,4’-DDT, 4,4’-DDT)

Dicofol

Dieldrin

Endosulfan (I and II)

Endrin

Heptachlor epoxidea

Hexachlorobenzene

Lindane (-hexachlorocyclohexane; -HCH)b

Mirexc

Toxaphene

Organophosphate Pesticides

Chlorpyrifos

Diazinon

Disulfoton

Ethion

Terbufos

Chlorophenoxy Herbicides

Oxyfluorfen

PAHsd

PCBs

Total PCBse

 (sum of PCB cogeners or Aroclor

equivalents)

Dioxins/furansf,g

PAHs = Polycyclic aromatic hydrocarbons; PCBs = Polychlorinated biphenyls; DDT = p,p’-dichlorodiphenyl

trichloroethane; DDE = p,p’-dichlorodiphenyl dichloroethylene; and DDD = dichlorodiphenyldichloro ethane.

a Heptachlor epoxide is not a pesticide but is a metabolite of two pesticides, heptachlor and chlordane. b Also known as -benzene hexachloride (-BHC). c Mirex should be regarded primarily as a regional target analyte in the Southeast and Great Lakes states, unless

historic tissue, sediment, or discharge data indicate the likelihood of its presence in other areas. d It is recommended that tissue samples be analyzed for benzo[a]pyrene, and 14 other PAHs and that the order-ofmagnitude relative potencies given for these PAHs be used to calculate a potency equivalency concentration

(PEC) for each sample for comparison with the recommended SVs for benzo[a]pyrene (see Section 5.3.2.5). e Analysis of total PCBs (as the sum of Aroclors or PCB congeners is recommended for conducting human health

risk assessments for total PCBs (see Sections 4.3.6 and 5.3.2.6). A standard method for Aroclor analysis is

available (EPA Method 608). A standard method for congener analysis (EPA Method 1668) is currently under

development; however, it has not been finalized. States that currently do congener-specific PCB analysis should

continue to do so and other states are encouraged to develop the capability to conduct PCB congener analysis. 

When standard methods for congener analysis are verified and peer reviewed, the Office of Water will evaluate the

use of these methods. f Note: The EPA Office of Research and Development is currently reassessing the human health effects of dioxins/

furans. g It is recommended that the 17 2,3,7,8-substituted tetra- through octa-chlorinated dibenzo- p-dioxins (PCDDs) and

dibenzofurans (PCDFs) and 12 dioxin-like PCBs be determined and a toxicity-weighted total concentration

calculated for each sample (Van den Berg et al., 1998) (see Sections 4.3.7, 5.3.2.6, and 5.3.2.7). 

2. Pesticides with active registrations—The EPA Office of Pesticide Programs

(OPP) Fate One Liners Database (U.S. EPA, 1993a) containing information

for more than 900 registered pesticides was reviewed to identify pesticides

and herbicides with active registrations that met four criteria. The screening

criteria used were 

• Oral toxicity, Class I or II

• Bioconcentration factor greater than 300

• Half-life value of 30 days or more

• Initial use application profile.
4. TARGET ANALYTES

4-3

At the time of this review, complete environmental fate information was

available for only about half of the registered pesticides. As more data

become available, additional pesticides will be evaluated for possible inclusion

on the target analyte list.

Use of the OPP database was necessary because many pesticides and

herbicides with active registrations have not been monitored extensively either

in national or state fish contaminant monitoring programs.

3. Contaminants that have triggered states to issue fish and shellfish

consumption advisories or bans—The database, National Listing of State

Fish and Shellfish Consumption Advisories and Bans (RTI, 1993), was

reviewed to identify specific chemical contaminants that have triggered

issuance of consumption advisories by the states. As shown in Table 4-2,

four contaminants (PCBs, mercury, chlordane, and dioxins/furans) triggered

advisories in the largest number of states in 1993. As a comparison, the

number of states issuing advisories for each pollutant in 1998 has also been

presented while the total number of states issuing advisories for most

pollutants generally has increased, the number of states issuing advisories for

two major pollutants, chlordane and dioxin, has decreased over the past

5 years.

4. Published literature on the chemistry and health effects of potential

contaminants—The physical, chemical, and toxicologic factors considered

to be of particular importance in developing the recommended target analyte

list were

• Oral toxicity

• Potential of the analyte to bioaccumulate

• Prevalence and persistence of the analyte in the environment

• Biochemical fate of the analyte in fish and shellfish

• Human health risk of exposure to the analyte via consumption of

contaminated fish and shellfish

• Analytical feasibility.

Final selection of contaminants by the EPA 1993 Workgroup for the recommended target analyte list (Table 4-1) was based on their frequency of inclusion in

national monitoring programs, on the number of states issuing consumption

advisories for them in 1993 (Table 4-2), and on their origins, chemistry, potential

to bioaccumulate, estimated human health risk, and feasibility of analysis.

Primary consideration was also given to the recommendations of the Committee

on Evaluation of the Safety of Fishery Products, published in Seafood Safety

(NAS, 1991).

4.2 SELECTION AND PRIORITIZATION OF TARGET ANALYTES

The decision to conduct a fish tissue monitoring study is normally the result of the

discovery of specific contaminants during water quality or sediment studies and/or
4. TARGET ANALYTES

4-4

Table 4-2. Contaminants Resulting in Fish and Shellfish Advisories

Contaminant

Number of states issuing advisories

1993 1998

Metals

Arsenic (total)

Cadmium

Chromium

Copper

Lead

Mercury

Selenium

Tributyltin

Zinc

Organometallics

Unidentified metals

1

2

1

1

4

29

5

1

1

1

3

3

3

1

1

5

40

5

0

1

1

1

Pesticides

Chlordane

DDT and metabolites

Dieldrin

Heptachlor epoxide

Hexachlorobenzene

Kepone

Mirex

Photomirex

Toxaphene

Unidentified pesticides

24

9

3

1

2

1

3

1

2

2

22

12

6

1

2

1

3

0

4

2

Polycyclic aromatic hydrocarbons (PAHs) 3 4

Polychlorinated biphenyls (PCBs) 32 36

Dioxins/furans 20 19

Other chlorinated organics

Dichlorobenzene

Hexachlorobutadiene

Pentachlorobenzene

Pentachlorophenol

Tetrachlorobenzene

Tetrachloroethane

1

1

1

1

2

1

1

1

0

2

0

0

Others

Creosote

Gasoline

Multiple pollutants

Phthalate esters

Polybrominated biphenyls (PBBs)

Unspecified pollutants

2

1

2

1

1

3

2

1

1

0

1

0

Sources: RTI, 1993; U.S. EPA, 1999c.

the identification of pollutant sources in waters routinely used by recreational or

subsistence fishers. EPA recognizes that measuring all 25 target analytes in fish

tissues collected at all state monitoring sites is expensive and that cost is an

important consideration that states must evaluate in designing and implementing
4. TARGET ANALYTES

4-5

their fish monitoring programs. Ideally, if resources are available to conduct

sampling and analysis of all 25 target analytes, the state should consider this

option because it provides the greatest amount of information for fishers in the

state on levels of contamination statewide. Also, this approach can better detect

the presence of those contaminants that are transported long distances from their

points of release (e.g., methylmercury, dioxins/furans, toxaphene), often outside

the state’s borders, and contaminate relatively pristine areas devoid of any

obvious pollutant sources. 

If the cost of this approach is prohibitive, however, the state may wish to use a

watershed-based approach as a way to reduce sampling and analysis costs

(Table 4-3). The selection and prioritization recommendations discussed below

are watershed-based and take into consideration land use categories (rural,

agricultural, suburban/urban, and industrial) as well as geological characteristics,

regional differences, and national pollution trends. Land use patterns (both

current and historic) are often the most important factors in deciding what

analytes to select for analysis. The watershed-based approach gives the highest

priority (XXX) to analysis of contaminants that are widely dispersed nationally and

relatively inexpensive to analyze, such as mercury. This approach gives a lower

priority (X) to monitoring organochlorine pesticides (e.g., chlordane, DDT, and

dieldrin) at rural and suburban sites, but a higher priority (XX) to monitoring these

same chemicals in agricultural watersheds where their use has been extensive

or in industrial watersheds where they may have been released during

manufacturing, formulation, packaging, or disposal. Because of the very high cost

of analysis for some contaminants (e.g., PCBs and dioxins/furans and dioxin-like

PCBs), this watershed approach also allows money for these analyses to be

directed toward analysis primarily in suburban/urban and industrial watersheds

where sources either from historic manufacturing or historic and/or current

practices (combustion or incineration sources) have been identified or where

water and/or sediment data in the watershed have detected these chemicals at

elevated concentrations. 

States should use all available environmental data and their best scientific

judgment when developing their fish monitoring programs. Using the watershed

approach gives states the flexibility to tailor their sampling and analysis programs

to obtain needed information as cost-effectively as possible by directing limited

resources to obtaining information on contaminant levels most likely to be found

in fish tissue at a given site. To be most effective, states need to recognize and

carefully evaluate all existing data when assessing which target analytes to

monitor at a particular site. States should include any of the recommended EPA

target analytes and any additional target analytes in their screening programs

when site-specific information (e.g., tissue, water, or sediment data; discharge

monitoring data from municipal and industrial sources; or pesticide use data)

suggests that these contaminants may be present at levels of concern for human

health. 
4. TARGET ANALYTES

4-6

Table 4-3. Selection and Prioritization of Target Analytes by

Watershed Type

Analyte

RuralAgriculturalSuburban/UrbanIndustrial

Sources/Uses

Metals

Arsenic XXa Xa,b Xa,b XXb Naturally occurring as a sulfide in mineral ores; fossil fuel

combustion; mining/smelting; wood preservative;

insecticide, herbicide, and algacide; hazardous waste site

leachate

Cadmium XXa

 Xa,b Xa,b XXb Smelting/mining; surface mine drainage; uses in paints,

alloys, batteries, plastics, pesticides, herbicides; waste

disposal operations.

Mercury XXXc XXXc

 XXXc XXXc Naturally occurring; atmospheric transport from fossil fuel

combustion; mining/smelting; chlorine alkali production;

historic use in pulp and paper and paints; Hazardous

waste site leachate; statewide freshwater and/or coastal

advisories in 15 states

Selenium XXa Xa Xa XXd Naturally occurring in west and southwest soils;

emissions from fossil fuel combustion; leachate from coal

fly ash disposal areas 

Tributyltin Xd XXd Shipyards and marinas; uses in antifouling paint, cooling

tower disinfectants, wood preservatives, pulp and paper

industry, and textile mills.

Organochlorine

Pesticides

Chlordane XXb Xb XXb Domestic termite control; pesticide manufacturing/

packaging/formulation sites 

DDT XXb Xb XXb Broad spectrum pesticide use; pesticide manufacturing/

packaging/formulation sites

Dicofole XXb XXb Miticide/pesticide for cotton, apples, and citrus primarily in

FL and CA; lesser use in turf, ornamentals, pears,

apricots, and cherries; pesticide manufacturing/

packaging/formulation sites

Dieldrin XXb Xb XXb Broad spectrum pesticide for termites/soil insects and for

cotton, corn, and citrus; pesticide manufacturing/

packaging/formulation sites

Endosulfane XXb XXb Noncontact insecticide for seed and soil treatments;

pesticide manufacturing/packaging/formulation sites

Endrin XXb XXb Broad spectrum pesticide; pesticide manufacturing/

packaging/formulation sites

Heptachlor epoxide XXb Xb XXb Degradation product of heptachlor used as a contact and

ingested soil insecticide for termites and household

pesticide and chlordane also used as a termiticide;

pesticide manufacturing/packaging/formulation sites for

heptachlor and chlordane
Table 4-3. (continued)

4. TARGET ANALYTES

Analyte

RuralAgriculturalSuburban/UrbanIndustrial

Sources/Uses

4-7

Hexachlorobenzene XXb XXb Fungicide used as seed protectant, used as chemical

intermediate in production of many other organochlorine

pesticides; pesticide manufacturing/packaging/formulation

sites for a wide variety of organochlorine pesticides

Lindanee XXb Xb XXb Seed and soil treatments for tobacco; foliage applications

for fruit and nut trees and vegetables; wood preservative. 

pesticide manufacturing/packaging/formulation sites

Mirex XXb Xb XXb Used extensively in Southeast and Gulf Coast states

against fire ants; used in fire retardants and plastic

polymerizer; pesticide manufacturing/packaging/

formulation sites

Toxaphene XXb XXb Insecticide for cotton; piscicide for rough fish; pesticide

manufacturing/packaging/formulation sites

Organophosphate

Pesticides

Chlorpyrifose XXb Xb XXb Widely used on cotton, peanuts, and sorghum as well as

fruits and vegetables; domestic household insecticide

with lawn and garden applications. Use applications will

change by the end of 2001. All residential use will end as

will use on tomatoes. Use on apples and grapes will be

greatly reduced (U.S. EPA, 2000b). Used as a termiticide

in California; pesticide manufacturing/packaging/

formulation sites

Diazinone XXb Xb XXb Widely used on a broad variety of fruits and vegetables,

field crops, and pastureland; domestic household

insecticide used for lawn and garden applications;

pesticide manufacturing/packaging/formulation sites

Disulfotone XXb XXb Widely used as a side dressing, broadcast, and foliar

spray and as a seed dressing; pesticide manufacturing/

packaging/formulation sites

Ethione XXb Xb XXb Major use on citrus, fruit and nut trees, and vegetables.

Domestic outdoor use around homes and lawns;

pesticide manufacturing/packaging/formulation sites

Terbufose XXb XXb Used principally on corn, sugar beets, and grain sorghum;

pesticide manufacturing/packaging/formulation sites

Chlorophenoxy

Herbicides

Oxyfluorfene XXb XXb Widely used to control grass and weeds in corn, cotton,

soybeans, fruit and nut trees, and ornamental crops;

pesticide manufacturing/packaging/formulation sites
Table 4-3. (continued)

4. TARGET ANALYTES

Analyte

RuralAgriculturalSuburban/UrbanIndustrial

Sources/Uses

4-8

Polycyclic Aromatic

Hydrocarbons (PAHS)

Xd Xd Components of crude and refined petroleum and coal

products; waste incineration, wood preservatives,

creosote, coal tar, coal coking, urban runoff from asphalt,

automobile tires and exhaust emissions, and petroleum

spills; coal gasification sites, and petroleum refineries.

Polychlorinated

Biphenyls (PCBs)

Xd

 Xd Produced as Arochlors for use as dielectric fluid in

electrical transformers and as hydraulic fluid; leachate

from land fills and Superfund sites.

Dioxins and

Dibenzofurans

Xd Xd Industrial sites including bleached kraft paper mills,

facilities handling 2,4,5-trichlorophenoxyacetic acid

(2,4,5,-T), 2,4,5-trichlorophenol (2,4,5-TCP), silvex,

hexachlorobenzene, pentachlorophenol, and PCBs.;

Industrial and municipal combustors and incinerators

a Tissue residue analysis is recommended if geologic characteristics suggest potential for elevated metal concentrations

in water or sediment or if sources are identified in the watershed suggesting the presence of this target analyte at the

sampling site. b Tissue residue analysis is recommended if use application of this pesticide has been reported in the watershed either

from historic or current use data, if sources like pesticide production/packaging/formulation facilities exist in the

watershed, or if the state has water and/or sediment data indicating the presence of this target analyte at the sampling

site. c Tissue residue analysis is highly recommended at all sites. d Tissue residue analysis is recommended if sources as described in Sources/Uses column are identified in

suburban/urban or industrial watershed or the state has water and/or sediment data indicating the presence of this

analyte at the sampling site. e Pesticide with currently active registration

X = Analysis for target analyte should be considered if water and or sediment analysis results detect the target

analyte or if historic or current use information provide evidence for the potential presence of this target

analyte in the watershed.

XX = Analysis for target analyte is recommended for this land use type if historic or current use information provides

evidence of the potential presence of this target analyte in the watershed. 

XXX = Analysis for target analyte is highly recommended at all stations in all watershed types.

Rural. The major analytes of concern in rural waterbodies (i.e., watersheds with

no past or current urban/suburban, industrial, or agricultural uses) are the metals,

including arsenic, cadmium, mercury, and selenium. Weathering processes in

certain geologic areas can result in elevated levels of arsenic, cadmium, mercury,

and selenium in water and sediments. State agencies should also be aware of

past land use patterns in what are now considered rural areas of their states. For

example, abandoned mining sites may be a source of metal contamination via

leaching from mine drainage or slag piles. Large areas east of the Appalachians

were agricultural watersheds during the early to mid twentieth century. While

some of this agriculture land is now suburban/urban in its use, other areas,

particularly in the South, are reverting to forests that might at first glance be

classified as rural use. Arsenic compounds were used as pesticides in the early
4. TARGET ANALYTES

4-9

1900s, and, along with organochlorine pesticides, may still be present in farmland

abandoned after the 1940s. States should also be aware that mercury has been

identified in fish collected from what would be classified as rural or pristine areas

of the Great Lakes basins and waterbodies in the northeastern and southeastern

states remote from any obvious point sources of pollution. Mercury contamination

in these areas seems to be facilitated through the atmospheric transport of this

metal. Because mercury is the target analyte that has triggered issuance of the

largest number of advisories in the United States (nearly 68 percent of all

advisories nationwide) and because of the relatively low cost of chemical analysis

for this analyte, EPA recommends that this metal be monitored at all rural sites,

especially those where little or no monitoring data are available. 

Depending on site-specific conditions and considerations, states may opt to

analyze for mercury as well as a suite of other heavy metals that can be analyzed

as a group at relatively low cost. The only target analyte metal that should not be

analyzed for routinely in rural areas without other supporting data is tributyltin,

which is typically found near boatyards and marinas or near wood preservative

production facilities. States may include any of the recommended EPA target

analytes and any additional target analytes in their screening programs when sitespecific information on a rural watershed suggests that these contaminants may

be present at levels of concern for human health.

Agricultural. The major analytes of concern in agricultural waterbodies (i.e.,

watersheds where past or current land use is dominated by agriculture) are the

organochlorine and organophosphate pesticides and the chlorophenoxy herbicide,

oxyfluorfen. These analytes fall into two categories, those with inactive registrations (i.e. banned or withdrawn from the market) and those with active

registrations (endosulfan, lindane, dicofol, chlorpyrifos, diazinon, terbufos, ethion,

disulfoton, and oxyfluorfen). Although use of some of the organochlorine

pesticides was terminated more than 20 years ago in the United States (e.g.,

DDT, dieldrin, endrin, and mirex) , these compounds still need to be monitored.

Many of the organochlorine pesticides that are now banned were used in large

quantities for over a decade and are still present in high concentrations at some

sites. On a nationwide basis, chlordane and DDT, for example, are responsible

for 3 and 1 percent, respectively, of the advisories currently in effect. For the

pesticides with active registrations, use and rate application information

maintained by the state’s Department of Agriculture should be reviewed to identify

watersheds where these pesticides are currently used and are likely to be present

in aquatic systems as a result of agricultural runoff or drift. Unlike many of the

historically used organochlorine pesticides, the pesticides in current use degrade

relatively rapidly in the environment. In addition, federal regulations are in effect

that set maximum application rates and minimize use near waterbodies. At the

time of this writing, no fish consumption advisories for these analytes have yet

been issued; however, state agencies should be aware of special circumstances

that could result in accumulation in fish. In addition to accidental spills and

misapplication, heavy and repeated rainfall shortly after application may wash

these pesticides into streams. Signs of pesticide pollution may include erratic

swimming behavior in fish as well as fish kills.
4. TARGET ANALYTES

4-10

It is also important to note that pesticide uses and labels may change over time.

All pesticides with active registrations are currently being reviewed by EPA under

provisions of the Food Quality Protection Act of 1996. The state agency

responsible for designing the fish contaminant monitoring program should be

aware of all historic and current uses of each pesticide within its state, including

the watersheds, application rates, and acreage where the pesticide has been or

currently is applied to ensure that all potentially contaminated sites are included

in the sampling plan. Because mercury contamination seems to be facilitated

through atmospheric transport, because it has triggered issuance of the largest

number of U.S. advisories, and because of the relatively low cost of chemical

analysis for this analyte, EPA recommends that this metal be monitored at all

agricultural sites, especially those for which little or no monitoring data are available. Additionally, states may also want to analyze for other metals (arsenic,

cadmium, and selenium). States may include any of the recommended EPA

target analytes and any additional target analytes in their screening programs

when site-specific information on an agricultural watershed suggests that these

contaminants may be present at levels of concern for human health. 

Suburban/Urban. Water and sediment quality are often regularly monitored in

suburban and urban areas, and selection of target analytes should be based on

these data when available. Some suburban watersheds of today were agricultural

watersheds during the early twentieth century. Arsenic compounds were widely

used as pesticides in the early 1900s, as were organochlorine pesticides. These

contaminants may still be present in farmland abandoned after the 1940s. As a

result of the rapid population growth in recent years, other suburban areas have

been built on former industrial sites, so historical information on land use should

be obtained by states whenever possible and reviewed carefully during the target

analyte selection process. 

Several of the organophosphates as well as organochlorine pesticides have had

wide use in control of pests around domestic structures as well as in lawn and

garden applications (see Table 4-3). Chlorpyrifos and diazinon are currently used

by pest control applicators and the general public (Robinson et al., 1994), and

diazinon has been reported at high concentrations in effluents from POTWs in

some suburban/urban areas (Amato et al., 1992; Burkhard and Jensen, 1993).

Historically, chlordane was used extensively in termite control around homes and

DDT was used as a general all-purpose insecticide. Nationally, chlordane and

DDT are responsible for 3 and 1 percent, respectively, of the advisories currently

in effect, and their use within suburban/urban watersheds should be considered

as should the use of any of the pesticides registered for use around domestic

structures or in lawn and garden applications. Depending on the proximity of

some suburban/urban sites to industrial areas, states may also wish to review

historic or current information on production sites associated with any of the

pesticides, PAHs, PCBs, and dioxin/furans. Because of the historic and current

uses of mercury in a variety of industrial processes, because it has triggered

issuance of the largest number of U.S. advisories, and because of the relatively

low cost of chemical analysis, EPA recommends that this metal be monitored at

all surburban/urban sites, especially those where either little or no monitoring data
4. TARGET ANALYTES

4-11

are available. States should include any of the recommended EPA target

analytes and any additional target analytes in their screening programs when sitespecific information on a suburban/urban watershed suggests that these

contaminants may be present at levels of concern for human health.

Industrial. All of the recommended target analytes can enter waterbodies

through releases from industrial processes, Superfund sites, or landfills. Often

water and sediment data are available to help guide the selection of the target

analytes that should be given high priority with respect to analysis. Selection of

analytes for analysis in industrial watersheds should be guided by knowledge of

the type of industrial production that has existed in the past or is currently present

in the watershed. Historical information is particularly important since potential

contaminants may still be present at abandoned industrial sites or contained in

sediments in receiving waterbodies. Sources of these target analytes are listed

in Section 4.3, which contains the individual target analyte profiles and descriptions of the types of industries that may contribute to releases of these specific

pollutants. Again, the states should review all existing water and sediment quality

data available before selecting the specific target analytes for analysis at each

site. Because of the historic and current uses of mercury in a variety of industrial

processes, because it has triggered issuance of the largest number of U.S.

advisories, and because of the relatively low cost of chemical analysis, EPA

recommends that this metal be monitored at all industrial sites, especially those

where little or no monitoring data are available. The other metals, including

tributyltin, should also be considered for analysis based on existence of industrial

production facilities, waste disposal facilities (e.g., Superfund or hazardous waste

sites, and landfills), or shipyards where these target analytes may have been

released to the environment. With respect to the pesticides, sites of production,

formulation, and packaging facilities can all potentially be sites for release of these

contaminants into the surrounding environment. Petroleum refining and coal

gasification and processing facilities can also be sites for discharges of PAHs.

PCBs can be released from historic landfills where PCB-containing equipment

was disposed of or from sites of historic PCB production or use. Dioxins and

dibenzofurans are likely to be found in proximity to historic or current industrial

sites such as bleached kraft paper mills or production facilities for 2,4,5-

trichlorophenoxyacetic acid (2,4,5-T), 2,4,5-trichlorophenol (2,4,5-TCP), and/or

silvex and medical, municipal, or industrial combustors or incinerators. States

should include any of the recommended EPA target analytes and any additional

target analytes in their screening programs when site-specific information on an

industrial watershed suggests that these contaminants may be present at levels

of concern for human health.

Specific factors that have been considered in the selection of the recommended

25 target analytes and sources for their release into the environment are

summarized in the next section. Chemical pollutants that are currently under

review by EPA’s Office of Water for inclusion as recommended target analytes are

discussed in Section 4.4. 
4. TARGET ANALYTES

4-12

4.3 TARGET ANALYTE PROFILES

4.3.1 Metals

Five metals—arsenic, cadmium, mercury, selenium, and tributyltin—are recommended as target analytes in screening studies. Arsenic, cadmium, and mercury

have been included in at least five of the eight major fish contaminant monitoring

programs reviewed by the 1993 Workgroup (see Appendix E). It should be noted,

however, that with respect to arsenic, all monitoring programs measured total

arsenic rather than inorganic arsenic. Selenium was monitored in four national

monitoring programs. Tributyltin, a constituent in antifouling paints was not

recommended for analysis in any of the national programs evaluated by the 1993

Workgroup. As of 1993, fish consumption advisories were in effect for arsenic,

cadmium, mercury, selenium, and tributyltin in 1, 2, 29, 5, and 1 states,

respectively (Table 4-2). As of 1998, fish advisories were in effect for arsenic,

cadmium, mercury, and selenium in 3, 3, 40, and 11 states, respectively. No

states had active advisories for tributyltin (U.S. EPA, 1999c). Also, with the

exception of tributyltin, these metals have been identified as having the greatest

potential toxicity resulting from ingestion of contaminated fish and shellfish (NAS,

1991).

4.3.1.1 Arsenic—

Arsenic is the twentieth most abundant element in the earth’s crust and naturally

occurs as a sulfide in a variety of mineral ores containing copper, lead, iron,

nickel, cobalt, and other metals (Eisler, 1988; Merck Index, 1989; Woolson, 1975).

Arsenic is released naturally to the atmosphere from volcanic eruptions and forest

fires (Walsh et al., 1979) and to water via natural weathering processes (U.S.

EPA, 1982b). Arsenic also has several major anthropogenic sources including

industrial emissions from coal-burning electric generating facilities, releases, as

a byproduct of nonferrous metal (gold, silver, copper, lead, uranium, and zinc)

mining and smelting operations (Eisler, 1988; May and McKinney, 1981; NAS,

1977), releases associated with its production and use as a wood preservative

(primarily as arsenic trioxide), and application as an insecticide, herbicide,

algicide, and growth stimulant for plants and animals (Appendix F) (Eisler, 1988).

Arsenic releases are also associated with leaching at hazardous waste disposal

sites and discharges from sewage treatment facilities. Arsenic trioxide is the

arsenic compound of chief commercial importance (U.S. EPA, 1982b) and was

produced in the United States until 1985 at the ASARCO smelter near Tacoma,

Washington. Arsenic is no longer produced commercially within the United States

in any significant quantities, but arsenic compounds are imported into the United

States primarily for use in various wood preservative and pesticide formulations.

The toxicity of arsenicals is highly dependent upon the nature of the compounds,

and particularly upon the valency state of the arsenic atom (Frost, 1967; Penrose,

1974; Vallee et al., 1960). Typically, compounds containing trivalent (+3) arsenic

are much more toxic than those containing pentavalent (+5) arsenic. The valency

of the arsenic atom is a more important factor in determining toxicity than the
4. TARGET ANALYTES

4-13

organic or inorganic nature of the arsenic-containing compound (Edmonds and

Francesconi, 1993). With respect to inorganic arsenic compounds, salts of

arsenic acid (arsenates) with arsenic in the pentavalent state are less toxic than

arsenite compounds with arsenic in the trivalent state (Penrose, 1974). Because

some reduction of arsenate (pentavalent arsenic) to arsenite (trivalent arsenic)

might occur in the mammalian body (Vahter and Envall, 1983), it would be unwise

to disregard the possible toxicity of inorganic arsenic ingested in either valency

state (Edmonds and Francesconi, 1993).

Seafood is a major source of trace amounts of arsenic in the human diet.

However, arsenic in the edible parts of fish and shellfish is predominantly present

as the arsenic-containing organic compound arsenobetaine (Cullen and Reimer,

1989; Edmonds and Francesconi, 1987a; NAS, 1991). Arsenobetaine is a stable

compound containing a pentavalent arsenic atom, which has been shown to be

metabolically inert and nontoxic in a number of studies (Cannon et al., 1983; Bos

et al., 1985; Kaise et al., 1985; Sabbioni et al., 1991; Vahter et al., 1983) and is

not generally considered a threat to human health (ATSDR, 1998a). Inorganic

arsenic, although a minor component of the total arsenic content of fish and

shellfish when compared to arsenobetaine, presents potential toxicity problems.

To the degree that inorganic forms of arsenic are either present in seafood or,

upon consumption, may be produced as metabolites of organic arsenic

compounds in seafood, some human health risk, although small, would be

expected (NAS, 1991).

Inorganic arsenic is very toxic to mammals and has been assigned to Toxicity

Class I based on oral toxicity tests (U.S. EPA, 1998d). Use of several arsenical

pesticides has been discontinued because of the health risks to animals and man.

Inorganic arsenic also has been classified as a human carcinogen (A), and longterm effects include dermal hyperkeratosis, dermal melanosis and carcinoma,

hepatomegaly, and peripheral neuropathy (IRIS, 1999) (Appendix G). 

Total arsenic (inclusive of both inorganic and organic forms) has been included

in five of the eight national monitoring programs evaluated by the 1993 Workgroup

(Appendix E). Arsenic and arsenic-containing organic compounds have not been

shown to bioaccumulate to any great extent in aquatic organisms (NAS, 1977).

Experimental evidence indicates that inorganic forms of both pentavalent and

trivalent arsenic bioaccumulate minimally in several species of finfish including

rainbow trout, bluegill, and fathead minnows (ASTER, 1999). A bioconcentration

factor (BCF) value of 350 was reported for the American oyster (Crassostrea

virginica) exposed to trivalent arsenic (Zaroogian and Hoffman, 1982).

In 1984 and 1985, the U.S. Fish and Wildlife Service collected 315 composite

samples of whole fish from 109 stations nationwide as part of the National

Contaminant Biomonitoring Program (Schmitt and Brumbaugh, 1990). The

authors reported the the maximum, geometric mean, and 85th percentile

concentrations for total arsenic were 1.5, 0.14, and 0.27 ppm (wet weight),

respectively. No information, however, was avaiIable on the percentage of

inorganic arsenic in the fish sampled in the NCBP study. Kidwell et al. (1995)
4. TARGET ANALYTES

4-14

conducted an analysis of total arsenic levels in bottom-feeding and predator fish

using the 1984-1985 data from the NCBP study. These authors reported that the

mean total arsenic tissue concentrations of 0.16 ± 0.23 ppm in bottom feeders

and 0.16 + 0.14 ppm in predator fish were not significantly different. 

Edmonds and Francesconi (1993) summarized existing data from studies

conducted outside the United States comparing concentrations of total arsenic,

organic arsenic, and inorganic arsenic in marine fish and shellfish. Inorganic

arsenic was found to represent from 0 to 44 percent of the total arsenic in marine

fish and shellfish species surveyed. Residue concentrations of inorganic arsenic

in the tissues typically ranged from 0 to 5.6 ppm (wet weight basis); but were

generally less than 0.5 ppm for most species. In a study of six species of

freshwater fish monitored as part of the Lower Columbia River study, inorganic

arsenic represented from 0.1 to 27 percent of the total arsenic, and tissue

residues of inorganic arsenic ranging from 0.001 to 0.047 ppm (wet weight) were

100 times lower than those reported for marine species (Tetra Tech, 1995).

In 1993, only one state (Oregon) had an advisory in effect for arsenic contamination (RTI, 1993). As of 1998, there were three advisories in effect in three states

(Louisiana, Oregon, and Washington) for this metal (U.S. EPA, 1999c). Because

it is the concentration of inorganic arsenic in fish and shellfish that poses the

greatest threat to human health, EPA recommends that total inorganic arsenic

(not total arsenic) be analyzed in contaminant monitoring programs. A chemical

analysis procedure for determining total inorganic arsenic residues in fish and

shellfish tissues is provided in Appendix H. Total inorganic arsenic should be

considered for inclusion in state fish and shellfish monitoring programs in areas

where it occurs in geologic formations, sites where mining or smelter operations

have occurred, or where its use is or has been extensive. States should contact

their appropriate state agencies to obtain information on the historic and current

uses of arsenic particularly as a wood preservative and in agricultural pesticides.

4.3.1.2 Cadmium—

Cadmium is commonly found in zinc, lead, and copper deposits (May and

McKinney, 1981). It is released into the environment from several anthropogenic

sources: smelting and refining of ores, electroplating, application of phosphate

fertilizers, surface mine drainage (Farag et al., 1998; U.S. EPA, 1978), and waste

disposal operations (municipal incineration and land application) (U.S. EPA,

1979a, 1987c). Cadmium is also used in the manufacture of paints, alloys,

batteries, and plastics and has been used in the control of moles and plant

diseases in lawns.

Cadmium is a cumulative human toxicant; it has been shown to cause renal

dysfunction and a degenerative bone disease, Itai-Itai, in Japanese populations

exposed via consumption of contaminated rice, fish, and water. Because

cadmium is retained in the kidney, older individuals (over 40-50 years of age)

typically have both the highest renal concentrations of cadmium and the highest

prevalence of renal dysfunction (U.S. EPA, 1979a). Cadmium is a known
4. TARGET ANALYTES

4-15

carcinogen in animals, and there is limited evidence of the carcinogenicity of

cadmium or cadmium compounds in humans. It has been classified by EPA as

a probable human carcinogen by inhalation (B1) (IRIS, 1999).

Cadmium has been found to bioaccumulate in fish and shellfish tissues in fresh

water (Schmitt and Brumbaugh, 1990) and in estuarine/marine waters (NOAA,

1987, 1989a) nationwide. In 1984 and 1985, the U.S. Fish and Wildlife Service

collected 315 composite samples of whole fish from 109 stations nationwide as

part of the NCBP (Schmitt and Brumbaugh, 1990). The authors reported the

maximum, geometric mean, and 85th percentile concentrations for cadmium were

0.22, 0.03, and 0.05 ppm (wet weight), respectively. In the NCBP study,

geometric mean concentrations of cadmium in freshwater fish were found to have

declined from 0.07 ppm in 1976 to 0.03 ppm in 1984 (Schmitt and Brumbaugh,

1990). This trend contradicts the general trend of increasing cadmium

concentrations in surface waters, which Smith et al. (1987) attribute to increasing

U.S. coal combustion (Schmitt and Brumbaugh, 1990). Kidwell et al. (1995)

conducted an analysis of cadmium concentrations in bottom-feeding and

predatory fish species using the 1984-1985 data from the NCBP study. These

authors found that mean cadmium tissue concentration (whole fish samples) of

0.04 ± 0.05 ppm in bottom feeders (e.g., carp, white sucker, and channel catfish)

was significantly higher than the mean cadmium tissue concentration of 0.01 ±

0.02 ppm found in predator fish (e.g., trout, walleye, largemouth bass).

In 1993, only two states (New York and Ohio) had issued fish advisories for

cadmium contamination (RTI, 1993). As of 1998, there were seven advisories in

effect in three states (Maine, New Jersey, and New York) for this heavy metal

(U.S. EPA, 1999c). Two of these states, New York and New Jersey, have issued

advisories for this metal in all of their marine coastal waters. Maine has a

statewide wildlife advisory in effect for cadmium in moose liver and kidney tissue

(U.S. EPA, 1999c). Cadmium should be considered for inclusion in all state fish

and shellfish contaminant monitoring programs in areas where it occurs in

geologic formations, where mining or smelter operations have occurred, or where

its use is or has been extensive.

4.3.1.3 Mercury—

A major source of atmospheric mercury is the natural degassing of the earth’s

crust, amounting to 2,700 to 6,000 tons per year (WHO, 1990) Primary points of

entry of mercury into the environment from anthropogenic sources include mining

and smelting, industrial processes including chlorine-alkali production facilities and

atmospheric deposition resulting from combustion of coal and other fossil fuels

and municipal and medical refuse incinerators (U.S. EPA, 1997c; Glass et al.,

1990). Primary industrial uses of mercury are in the manufacture of batteries,

vapor discharge lamps, rectifiers, fluorescent bulbs, switches, thermometers, and

industrial control instruments (May and McKinney, 1981), and these products

ultimately end up in landfills or incinerators. Mercury has also been used as a

slimicide in the pulp and paper industry, as an antifouling and mildew-proofing
4. TARGET ANALYTES

4-16

agent in paints, and as an antifungal seed dressing (ATSDR, 1998; Farm

Chemicals Handbook, 1989; Friberg and Vostal, 1972).

Although mercury use and losses from industrial processes in the United States

have been reduced significantly since the 1970s, mercury contamination

associated with increased fossil fuel combustion is of concern in some areas and

may pose more widespread contamination problems in the future. An estimated

5,000 tons of mercury per year is released into the environment from fossil fuel

burning (Klaassen et al., 1986). The best estimate of annual anthropogenic U.S.

emissions of mercury in 1994-1995 was 158 tons. Of this, about 87 percent was

released from combustion sources, including waste and fuel combustion. (U.S.

EPA, 1997). There is also increasing evidence of elevated mercury concentrations in areas where acid rain is believed to be a factor (NESCAUM, 1998;

Sheffy, 1987; Wiener, 1987). Volatilization from surfaces painted with mercurycontaining paints, both indoors and outdoors, may have been a significant source

in the past (Agocs et al., 1990; Sheffy, 1987). The United States estimated that

480,000 pounds of mercuric fungicides were used in paints and coatings in 1987

(NPCA, 1988). In July 1990, EPA announced an agreement with the National

Paint and Coatings Association to cancel all registrations for use of mercury or

mercury compounds in interior paints and coatings. In May 1991, the paint

industry voluntarily canceled all remaining registrations for mercury in exterior

paints.

Cycling of mercury in the environment is facilitated by the volatile character of its

metallic form and by bacterial transformation of metallic and inorganic forms to

stable alkyl mercury compounds, particularly in bottom sediments, which leads to

bioaccumulation of mercury (Wood, 1974). Practically all mercury in fish tissue

is in the form of methylmercury (Bache et al., 1971; Bloom, 1992; Kannan et al.,

1998; Spry and Wiener, 1991), which is toxic to humans (NAS, 1991; Tollefson,

1989), with the percentage of methylmercury to total mercury in the muscle tissue

increasing as the fish ages (Bache et al., 1971). Several studies have shown that

mercury concentrations in fish tissue generally increase with age, and therefore

size (length or weight), owing to methylmercury accumulation with increasing

duration of exposure (Driscoll et al., 1994; Jackson, 1990; Johnson, 1987; Lange

et al., 1993); however this relationship is not as strongly correlated in all

environmental situations or for all fish species (Goldstein et al., 1996; Neumann

et al., 1997). 

EPA has classified methylmercury as a Group C, possible human carcinogen,

based on inadequate data in humans and limited evidence in animals

(Appendix G). No persuasive evidence of increased carcinogenicity attributable

to methylmercury exposure was observed in three human studies; however,

interpretation of these studies was limited by poor study design and other

problems. Animal studies have shown significant increases in the incidences of

kidney tumors in male, but not in female, mice (IRIS, 1999). 

Both inorganic and organic forms of mercury are neurotoxicants. Fetuses

exposed to organic mercury have been found to be born mentally retarded and
4. TARGET ANALYTES

4-17

with symptoms similar to those of cerebral palsy (Marsh, 1987; U.S. EPA, 1997c).

Individuals exposed to mercury via long-term ingestion of mercury-contaminated

fish have been found to exhibit a wide range of symptoms, including numbness

of the extremities, tremors, spasms, personality and behavior changes, difficulty

in walking, deafness, blindness, and death (U.S. EPA, 1997c). Organomercury

compounds were the causative agents of Minamata Disease, a neurological

disorder reported in Japan during the 1950s among individuals consuming

contaminated fish and shellfish (Kurland et al., 1960), with infants exposed

prenatally found to be at significantly higher risk than adults. Another methylmercury poisoning incident involving fish and shellfish occurred in 1965 in Niigata,

Japan. A third methylmercury poisoning incident occurred in the late 1960s and

early 1970s in Iraq; however, this last incident was associated with the accidental

consumption of seed grain treated with organomercury fungicide (U.S. EPA,

1997c). The EPA is especially concerned about evidence that the fetus is at

increased risk of adverse neurological effects from exposure to methylmercury

(e.g., Marsh et al., 1987; Piotrowski and Inskip, 1981; Skerfving, 1988; WHO,

1976, 1990; U.S. EPA, 1997c).

The EPA has set an interim Reference Dose (RfD) for methylmercury of

0.1 g/kg-d (IRIS 1999). The National Academy of Sciences (NAS) conducted an

independent assessment of the interim RfD. They concluded “On the basis of its

evalution, the committee’s consensus is that the value of EPA’s current RfD for

methylmercury, 0.1 μg/kg per day, is a scientifically justifiable level for the

protection of public health”. However, the NAS recommended that the Iraqi study

no longer be used as the scientific basis for the RfD. In addition, the NAS

recommended that the developmental neurotoxic effects of methylmercury

reported in the Faroe Islands study should be used as the basis for the derivation

of the RfD.” (NAS, 2000)

Mercury has been found in both fish and shellfish from estuarine/marine (NOAA,

1987, 1989a) and fresh waters (Schmitt and Brumbaugh, 1990) at diverse

locations nationwide. In 1984 and 1985, the U.S. Fish and Wildlife Service

collected 315 composite samples of whole fish from 109 stations nationwide as

part of the National Contaminant Biomonitoring Program (NCBP) (Schmitt and

Brumbaugh, 1990). The authors reported that the maximum, geometric mean,

and 85th percentile concentrations for mercury were 0.37, 0.10, and 0.17 ppm (wet

weight), respectively. In contrast to cadmium and selenium, concentrations of

mercury in freshwater fish tissue did not decline between 1976 and 1984 (Schmitt

and Brumbaugh, 1990). Kidwell et al. (1995) conducted an analysis of mercury

levels in bottom-feeding and predator fish using the 1984-1985 data from the

NCBP study. These authors reported that the mean mercury tissue concentration

(whole fish samples) of 0.12 ± 0.08 ppm in predator fish (e.g., trout, walleye,

largemouth bass) was significantly higher than the mean tissue concentration of

0.08 + 0.006 ppm in bottom feeders (e.g., carp, white sucker, and channel

catfish). 

Mercury, the only metal analyzed as part of the EPA National Study of Chemical

Residues in Fish, was detected at 92 percent of 374 sites surveyed. Maximum,
4. TARGET ANALYTES

4-18

arithmetic mean, and median concentrations in fish tissue were 1.77, 0.26, and

0.17 ppm (wet weight), respectively (U.S. EPA, 1991h, 1992c, 1992d). Bahnick

et al. (1994) analyzed the NSCRF data by fish species and reported that mean

mercury concentrations in bottom feeders (whole body samples) were generally

lower than concentrations for predator fish (fillet samples). Carp, white sucker,

and channel catfish (bottom feeders) had average tissue concentrations of 0.11,

0.11, and 0.09 ppm, respectively. Largemouth bass, smallmouth bass, and

walleye (predator species) had average tissue concentrations of 0.46, 0.34, and

0.52 ppm, respectively (Bahnick et al., 1994). With regard to the source of the

mercury contamination, Bahnick et al. (1994) reported that the highest mean

concentration of mercury was detected in fish sampled near public treatment

works (0.59 ppm); however, background sites and sites near wood preserving

facitities exhibited the second (0.34 ppm) and third (0.31 ppm) highest mean

mercury concentrations. The authors also reported that most of the higher tissue

concentrations of mercury were detected in freshwater fish samples collected in

the Northeast.

Recently, the northeastern states and eastern Canadian provinces issued their

own mercury study, including a comprehensive analysis of mercury concentrations in a variety of freshwater sportfish (NESCAUM, 1998). This study

involved a large number of sampling sites, including remote lake sites that did not

receive point source discharges. Top-level piscivores (i.e., predator fish), such

as walleye, chain pickerel, and large and smallmouth bass, were typically found

to exhibit the highest concentrations, with mean tissue residues greater than 0.5

ppm and maximum residues exceeding 2 ppm. One largemouth bass sample

was found to contain 8.94 ppm of mercury, while a smallmouth bass sampled

contained 5 ppm. A summary of the range and the mean concentrations found

in eight species of sportfish sampled is shown in Table 4-4 (NESCAUM, 1998).

Table 4-4. Total Mercury and Methylmercury Concentrations in

Estuarine Fish from South Florida

Species

Mean mercury concentrationa

(ppm) and range

Mean methylmercurya

concentration (ppm) and range

Hardhead catfish 1.94 (0.44-4.64) 1.54 (0.18-4.42)

Gafftopsail catfish 3.0 (0.76-10.10) 1.86 (0.72-4.50)

Sand seatrout 2.41 (2.21-2.61) 2.04 (1.60-2.47)

Sand seaperch 0.48 (0.40-0.54) 0.42 (0.40-0.49)

Pinfish 0.54 (0.32-1.06) 0.44 (0.20-0.90)

White grunt 0.49 (0.28-1.03) 0.49 (0.31-0.99)

Lane snapper 0.57 (0.22-1.03) 0.58 (0.19-1.27)

Spot 0.29 (0.11-0.43) 0.24 (0.06-0.40)

a

Concentrations are in ppm (μg/g) wet weight basis.

Source: Kannan et al., 1998.
4. TARGET ANALYTES

4-19

EPA’s Office of Water also recently published results of a national survey of

mercury concentrations in fish (U.S. EPA, 1999d). This survey compiled state

data on tissue residue levels of mercury in fish analyzed by 39 states between

1990 and 1995. The range of mean mercury concentrations (ppm) for the nine

major fish species reported were as follows: largemouth bass, 0.001-8.94;

smallmouth bass, 0.008-3.34; walleye, 0.008-3.0; northern pike, 0.10-4.4; channel

catfish, 0.001-2.57; bluegill sunfish, 0.001-1.68; common carp, 0.001-1.8; white

sucker, 0.002-1.71; and yellow perch, 0.01-2.14. All mercury concentrations used

in the study were expressed on a wet weight and fillet basis. While the majority

of the finfish sampled were freshwater species, some estuarine and marine

species were also included; however, the report excluded all nonfish species such

as turtles, molluscs, and crustaceans. Although comparison of data between

states was difficult because of differences in sampling strategies (representative

versus targeted), differences in analytical procedures, and the fact that mercury

concentrations may vary with age of the fish, the analysis did indicate that both

the magnitude and variability of mercury concentrations were greater in higher

trophic level fish species. 

Another recent study was conducted to assess total mercury and methylmercury

concentrations in estuarine fish from south Florida coastal waters (Kannan et al.,

1998). The authors reported that concentrations of total mercury in fish muscle

tissue ranged between 0.03 and 2.22 ppm (mean: 0.31 ppm) (wet weight basis),

with methylmercury contributing 83 percent of the total mercury. The mean

concentrations and range of total mercury and methylmercury in muscle tissue of

different species collected from south Florida’s coastal waters are shown in

Table 4-4. 

In another study, methylmercury concentrations in muscle tissue of nine species

of sharks were analyzed from four different locations along the coast of Florida

(Hueter et al., 1995). Muscle tissue methylmercury concentrations averaged

0.88 ppm (wet weight) and ranged from 0.06 to 2.87 ppm, with 31 percent of the

samples tested exceeding 1 ppm. A positive correlation was found between

methylmercury concentration and the body length (size) of the shark, such that

sharks larger than 2 m in total length contained methylmercury concentrations

>1 ppm. Sharks collected off the southern and southwestern coastal areas

contained significantly higher concentrations than those caught in the northeast

coastal region (Cape Canaveral and north). Methylmercury concentrations were

highest in the Caribbean reef shark (Carcharhinus perezi). The two most

abundant shark species in the U.S. East Coast commercial shark fishery, the

sandbar (C. plumbeus) and blacktip (C. limbatus) sharks, are of special public

health concern. Although the mean methylmercury concentration in the sandbar

shark (0.77 ppm) was below the average for all sharks, sandbar shark tissues

contained up to 2.87 ppm methylmercury, and 20.9 percent of the sampled fish

exceeded 1 ppm. Of more concern is that 71.4 percent of the blacktip shark

samples (mean, 1.3 ppm) exceeded 1 ppm methylmercury. The authors suggest

that continued monitoring of methylmercury concentrations in various shark

species is warranted, since these fish are taken in both recreational and

commercial fisheries. Similarly, on the West Coast, Fairey et al. (1997) reported
4. TARGET ANALYTES

4-20

that the highest concentrations of mercury found in all of the fish species sampled

as part of a fish monitoring effort in the San Franscico Bay and Estuary were

detected in leopard shark muscle tissue (1.26 ppm wet weight basis).

In 1993, 898 fish advisories had been issued in 29 states as a result of mercury

contamination (see Figure 4-1). In particular, mercury was included in a large

number of the fish advisories in effect for lakes in Minnesota, Wisconsin, and

Michigan and for rivers and lakes in Florida (RTI, 1993). As of 1998, 1,931

advisories had been issued in 40 states for this metal, and mercury is responsible

for more than 68 percent of all fish advisories issued in the United States. In

addition, 10 states have statewide advisories in effect for mercury in freshwater

lakes and/or rivers and 5 Gulf Coast states have statewide mercury advisories in

effect for their coastal marine waters (U.S. EPA, 1999c).

Because of its widespread occurrence in fish across the United States, mercury

should be monitored in all state fish and shellfish contaminant monitoring

programs at all stations. Only one national program reviewed by the 1993

Workgroup—EPA 301(h) monitoring program—recommended analyzing

specifically for methylmercury; however, six programs recommended analyzing

for total mercury (Appendix E). Because of the higher cost of methylmercury

analysis two to three times greater than for total mercury analysis). EPA

recommends that total mercury be determined in state fish contaminant

monitoring programs and the conservative assumption be made that all mercury

is present as methylmercury so as to be most protective of human health. It

should be noted that Bache et al. (1971) analyzed methylmercury concentrations

in lake trout of known ages and found that methylmercury concentration and the

ratio of methylmercury to total mercury increased with age. Relative proportions

of methylmercury in fish varied between 30 and 100 percent, with methylmercury

concentrations lower than 80 percent occurring in fish 3 years of age or younger.

Thus, when high concentrations of total mercury are detected, and if resources

are sufficient, states may wish to repeat sampling and obtain more specific

information on actual concentrations of methylmercury in various age or size

classes of fish.

4.3.1.4 Selenium—

Selenium is a natural component of many soils, particularly in the west and

southwest regions of the United States (NAS, 1991). It enters the environment

primarily via emissions from oil and coal combustion (May and McKinney, 1981;

Pillay et al., 1969). Selenium is an essential nutrient but is toxic to both humans

and animals at high concentrations (NAS, 1991). Long-term adverse effects from

ingestion by humans have not been studied thoroughly. EPA has determined that

the evidence of carcinogenicity of selenium in both humans and animals is

inadequate and, therefore, has assigned this metal a D carcinogenicity classification (IRIS, 1999).

Selenium is frequently detected in ground and surface waters in most regions of

the United States and has been detected in marine fish and shellfish (NOAA, 
4. TARGET ANALYTES

4-21

Figure 4-1. States issuing fish and shellfish advisories for mercury.
4. TARGET ANALYTES

4-22

1987, 1989a) and in freshwater fish (Schmitt and Brumbaugh, 1990) from several

areas nationwide. In 1984 and 1985, the U.S. Fish and Wildlife Service collected

315 composite samples of whole fish from 109 stations nationwide as part of the

National Contaminant Biomonitoring Program (Schmitt and Brumbaugh, 1990).

The authors reported the maximum, geometric mean, and 85th percentile

concentrations for selenium were 2.30, 0.42, and 0.73 ppm (wet weight), respectively. Kidwell et al. (1995) conducted an analysis of selenium concentrations in

bottom-feeding and predator fish using the 1984-1985 data from the NCBP study.

Mean selenium tissue concentrations (whole fish samples) were not significantly

different in bottom feeders (0.50 ± 0.41 ppm) as compared to predator fish (0.50

± 0.42 ppm). Like cadmium, concentrations of selenium declined in fish tissues

between 1976 and 1984 (Schmitt and Brumbaugh, 1990). 

In a more recent study (May 1993 to January 1994), selenium concentrations in

the tissues of fish from the Pigeon River and Pigeon Lake in Michigan were

examined. Mean selenium concentrations in white sucker fillets were 0.49 ± 0.19,

1.8 ± 0.96, and 1.7 ± 0.80 ppm (wet weight) in samples taken from the Upper

Pigeon River, Lower Pigeon River, and Pigeon Lake, respectively. At these same

locations, northern pike fillets contained selenium concentrations of 0.88 ± 0.22,

1.1 ± 0.91, and 2.2 ± 0.90 ppm (wet weight), respectively (Besser et al., 1996).

This study was conducted to assess the potential hazard of selenium leaching

from a coal fly ash disposal area. 

Selenium was monitored in four national fish contaminant monitoring programs

reviewed by the EPA 1993 Workgroup (Appendix E). Definitive information

concerning the chemical forms of selenium found in fish and shellfish is not

available (NAS, 1976, 1991).

In 1993, five states (California, Colorado, North Carolina, Texas, and Utah) had

issued advisories for selenium contamination in fish (RTI, 1993). As of 1998,

there were 11 advisories in effect in these same five states for this heavy metal

(U.S. EPA, 1999c). These advisories include one wildlife advisory in Nevada for

selenium in several species of waterfowl. Selenium should be considered for

inclusion in all state fish and shellfish monitoring programs in areas where it

occurs in geologic formations (particularly in the western and southwestern states)

and near sites where oil or coal combustion currently occurs or historically has

occurred.

4.3.1.5 Tributyltin Compounds—

Tributyltin compounds belong to the organometallic family of tin compounds that

have been used as biocides, disinfectants, and antifoulants. Antifoulant paints

containing tributyltin compounds were first registered for use in the United States

in the early 1960s (Appendix F). Tributyltin compounds are used in paints applied

to boat and ship hulls as well as to crab pots, fishing nets, and buoys to retard the

growth of fouling organisms. These compounds were also registered for use as

wood preservatives, disinfectants, and biocides in cooling towers, pulp and paper

mills, breweries, leather processing facilities, and textile mills (U.S. EPA, 1988c).
4. TARGET ANALYTES

4-23

Tributyltin compounds are acutely toxic to aquatic organisms at concentrations

below 1 ppb and are chronically toxic to aquatic organisms at concentrations as

low as 0.002 ppb (U.S. EPA, 1988c). EPA initiated a Special Review of tributyltin

compounds used as antifoulants in January of 1986 based on concerns over its

adverse effects on nontarget aquatic species. Shortly thereafter the Organotin

Antifouling Paint Control Act (OAPCA) was enacted in June 1988, which

contained interim and permanent tributyltin use restrictions as well as

environmental monitoring, research, and reporting requirements. The Act

established interim release rate restrictions under which only tributyltin-containing

products that do not exceed an average daily release rate of 4 micrograms

organotin/cm2

-d can be sold or used. The OAPCA also contained a permanent

provision to prohibit the application of tributyltin antifouling paints to non-aluminum

vessels under 25 meters (82 feet) long (U.S. EPA, 1988c).

Tributyltin oxide appears to be toxic to animals, with oral LD50s ranging between

52 and 194 mg/kg (ATSDR, 1992; HSDB, 1999; WHO, 1999). Immunotoxicity

is the critical effect produced by chronic exposure to tributyltin. Insufficient data

are available to evaluate the carcinogenicity of tributyltin oxide compounds;

therefore, EPA has listed this compound in Group D (Appendix G) (IRIS, 1999).

Tributyltins have been found to bioaccumulate in fish, bivalve mollusks, and

crustaceans. Bioconcentration factors have been reported to range from 200 to

4,300 for finfish, from 2,000 to 6,000 for bivalves, and a BCF value of 4,400 was

reported for crustaceans (U.S. EPA, 1988c). Tributyltin used to control marine

fouling organisms in an aquaculture rearing pen has been found to bioaccumulate

in fish tissue (Short and Thrower, 1987a and 1987b). Tsuda et al. (1988) reported

a BCF value of 501 for tributyltin in carp (Cyprinus carpio) muscle tissue. Martin

et al. (1989) reported a similar BCF value of 406 for tributyltin in rainbow trout

(Salmo gairdneri) and Ward et al. (1981) reported a BCF value of 520 for the

sheepshead minnow (Cyprinodon variegatus). In an environmental monitoring

study conducted in England, a BCF value of 1,000 was reported for tributyltin in

seed oysters (Crassostrea gigas) (Ebdon et al., 1989).

Tributyltin was not monitored in any national fish contaminant monitoring program

evaluated by the EPA 1993 Workgroup (Appendix E). In 1993, only one state,

Oregon, had an advisory in effect for tributyltin contamination in shellfish (RTI,

1993). As of 1998, there were no active fish advisories in effect for tributyltin,

since the advisory in Oregon was rescinded (U.S. EPA, 1999c).

Tributyltin compounds should be considered for inclusion in all state fish and

shellfish contaminant monitoring programs, particularly in states with coastal

waters, states bordering the Great Lakes, or states with large rivers where large

ocean-going vessels are used for commerce. Tributyltin concentrations have been

reported to be highest in areas of heavy boating and shipping activities including

shipyards, drydocks, and marinas where tributyltin-containing antifouling paints

are often removed and reapplied. Before recoating, old paint containing tributyltin

residues is scraped from the vessel hull and these paint scrapings are sometimes

washed into the water adjacent to the boat or shipyard despite the tributyltin label
4. TARGET ANALYTES

4-24

prohibiting this practice (U.S. EPA, 1988c). Tributyltin should be considered for

inclusion in state fish and shellfish monitoring programs in areas where its use is

or has been extensive. States should contact their appropriate agencies to obtain

information on the historic and current uses of tributyltin, particularly with respect

to its uses in antifouling paints and wood preservatives.

4.3.2 Organochlorine Pesticides

The following organochlorine pesticides and metabolites are recommended as

target analytes in screening studies: total chlordane (sum of cis- and transchlordane, cis- and trans-nonachlor, and oxychlordane), total DDT (sum of 2,4’-

and 4,4’-homologues of DDT, DDD, and DDE), dicofol, dieldrin, endosulfan I and

II, endrin, heptachlor epoxide, hexachlorobenzene, lindane (-hexachlorocyclohexane), mirex, and toxaphene (see Appendix F). Mirex is of particular concern

in the Great Lakes states and the southeast states (NAS, 1991). All of these

compounds are neurotoxins and most are known or suspected human carcinogens (IRIS, 1999; Sax, 1984).

With the exception of endosulfan I and II, dicofol, and total DDT, each of the

pesticides on the recommended target analyte list (Table 4-1) had been included

in at least four major fish contaminant monitoring programs (Appendix E), and

seven of the compounds had triggered at least one state fish consumption

advisory in 1993 (Table 4-2). Although use of some of these pesticides has been

terminated or suspended within the United States for over 25 years (Appendix F),

these compounds still require long-term monitoring. Many of the organochlorine

pesticides that are now banned were used in large quantities for over a decade

and are still present in sediments at high concentrations. These organochlorine

pesticides are not easily degraded or metabolized and, therefore, persist in the

environment. These compounds are either insoluble or have relatively low

solubility in water, but are quite lipid-soluble. Because these compounds are not

readily metabolized or excreted from the body and are readily stored in fatty

tissues, they can bioaccumulate to high concentrations through aquatic food

chains to secondary consumers (e.g., fish, piscivorous birds, and mammals

including humans).

Pesticides may enter aquatic ecosystems from point source industrial discharges

or from nonpoint sources such as aerial drift and/or runoff from agricultural use

areas, leaching from landfills, or accidental spills or releases. Agricultural runoff

from crop and grazing lands is considered to be the major source of pesticides in

water, with industrial waste (effluents) from pesticide manufacturing the next most

common source (Li, 1975). Significant atmospheric transport of pesticides to

aquatic ecosystems can also result from aerial drift of pesticides, volatilization

from applications in terrestrial environments, and wind erosion of treated soil (Li,

1975). Once in water, pesticide residues may become adsorbed to suspended

material, deposited in bottom sediment, or absorbed by organisms in which they

are detoxified and eliminated or accumulated (Nimmo, 1985).
4. TARGET ANALYTES

4-25

The reader should note that three of the organochlorine pesticides still have active

registrations: endosulfan, lindane, and dicofol. These pesticides are much less

persistent in the environment and have a lower bioaccumulation potential than the

banned organochlorines. However, agricultural runoff particularly during the

period immediately after field application could result in significant levels of these

pesticides in fish and shellfish tissues. States should contact their appropriate

state agencies to obtain information on both the historic and current uses of these

pesticides.

4.3.2.1 Chlordane (Total)—

Chlordane is a multipurpose insecticide that has been used extensively in home

and agricultural applications in the United States for the control of termites and

many other insects (Appendix F). This pesticide is similar in chemical structure

to dieldrin, although less toxic (Toxicity Class II), and has been classified as a

probable human carcinogen (B2) by EPA (Appendix G) (IRIS, 1999; Worthing,

1991).

Although the last labeled use of chlordane as a termiticide was phased out in the

United States beginning in 1975, it has been monitored in seven national fish

contaminant programs evaluated by the EPA 1993 Workgroup (Appendix E) and

has been widely detected in freshwater fish (Schmitt et al., 1990) and in both

estuarine/marine finfish (NOAA, 1987) and marine bivalves (NOAA, 1989a) at

concentrations of human health concern. In 1984 and 1985, the U.S. Fish and

Wildlife Service collected 321 composite samples of whole fish from 112 stations

nationwide as part of the National Contaminant Biomonitoring Program (Schmitt

et al., 1990). These authors reported the maximum and geometric mean

concentrations for the five major degradation products of chlordane (cischlordane, trans-chlordane, cis-nonachlor, trans-nonachlor, and oxychlordane)

were 0.66 and 0.03 ppm, 0.35 and 0.02 ppm, 0.45 and 0.02 ppm, 1.00 and

0.30 ppm, and 0.29 and 0.01 ppm (wet weight), respectively. Kidwell et al. (1995)

conducted an analysis of all 1984-1985 data from the NCBP study on the major

constituents of chlordane (including cis- and trans-chlordane, cis- and transnonachlor, and oxychlordane) in bottom-feeding and predator fish species. The

authors reported there was no significant difference in residues in these two

trophic groups of fish except for concentrations of trans-chlordane, which were

significantly higher in the tissues of bottom feeders. Mean tissue concentrations

of cis- and trans-chlordane, cis- and trans-nonachlor, and oxychlordane were 0.03

± 0.06, 0.02 ± 0.04, 0.02 ± 0.04, 0.03 ± 0.01, and 0.01 ± 0.02 ppm, respectively,

for bottom feeders as compared to 0.02 ± 0.04, 0.01 ± 0.02, 0.02 ± 0.03, 0.03 ±

0.06, and 0.01 ± 0.01 ppm, respectively, for predator species (Kidwell et al.,

1995).

The cis- and trans-isomers of chlordane and cis- and trans-isomers of nonachlor,

which are primary constituents of technical-grade chlordane, and oxychlordane,

the major metabolite of chlordane, were also monitored as part of the EPA

National Study of Chemical Residues in Fish (U.S. EPA, 1992c, 1992d ). These

compounds were detected in fish tissue at the following percentage of the 362
4. TARGET ANALYTES

4-26

sites surveyed: cis-chlordane (64 percent), trans-chlordane (61 percent), cisnonachlor (35 percent), trans-nonachlor (77 percent), and oxychlordane (27

percent) (U.S. EPA, 1992c, 1992d). The maximum, arithmetic mean, and median

concentrations (wet weight) of cis-chlordane, trans-chlordane, cis-nonachlor,

trans-nonachlor, and oxychlordane are summarized in Table 4-5. Mean total

chlordane residues from the NSCRF study were highest in bottom feeders such

as carp (0.067 ppm), white sucker (0.018 ppm), and channel catfish (0.054 ppm)

as compared to predator fish such as largemouth bass (0.029 ppm), smallmouth

bass (0.004 ppb), and walleye (0.004 ppm) (Kuehl et al., 1994). 

In 1993, 120 fish advisories in 24 states had been issued as a result of chlordane

contamination (see Figure 4-2). As of 1998, there were 104 advisories in effect

in 22 states for this pesticide, and New York currently has a statewide advisory

for chlordane in all waterfowl (U.S. EPA, 1999c). Because of its extensive use in

termite control and its widespread detection in fish tissues, total chlordane (i.e.,

sum of cis- and trans-chlordane, cis- and trans-nonachlor, and oxychlordane)

should be considered for inclusion in all state fish and shellfish contaminant

monitoring programs (NAS, 1991). Monitoring sites in agricultural watersheds

should be reviewed to determine the application rate and acreage where chlordane was used historically. In suburban/urban watersheds, the degree of historic

use of chlordane as a termiticide around domestic structures should also be

evaluated. Sites in industrial watersheds should be reviewed to identify historic

sites of chlordane production, formulation, or packaging facilities.

4.3.2.2 DDT (Total)—

Although the use of DDT was terminated in the United States in 1972, DDT and

its DDE and DDD metabolites persist in the environment and are known to

bioaccumulate (Ware, 1978). DDT, DDD, and DDE have all been classified by

EPA as probable human carcinogens (B2) (Appendix G) (IRIS, 1999).

Table 4-5. Chlordane Constituent Concentrationsa Detected in the EPA

National Study of Chemical Residues in Fish

Chlordane

constituent or

metabolite Maximum Arithmetic mean Median

cis-Chlordane 0.378 0.021 0.004

trans-Chlordane 0.310 0.017 0.003

cis-Nonachlor 0.127 0.009 ND

trans-Nonachlor 0.477 0.031 0.009

Oxychlordane 0.243 0.005 ND

ND = Not detected.

a

Concentrations are in ppm (micrograms/g) on a wet weight basis.

Source: U.S. EPA, 1992c,1992d.
4. TARGET ANALYTES

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Figure 4-2. States issuing fish and shellfish advisories for chlordane.
4. TARGET ANALYTES

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DDT or its metabolites have been included as target analytes in as many as

seven major fish and shellfish monitoring programs (Appendix E) and contamination has been found to be widespread (NOAA, 1987, 1989a; Schmitt et al., 1990).

In 1984 and 1985, the U.S. Fish and Wildlife Service collected 321 composite

samples of whole fish from 112 stations nationwide as part of the National

Contaminant Biomonitoring Program (Schmitt et al., 1990). Maximum and

geometric mean tissue concentrations of DDT, DDE, and DDD in 1984 were 1.79

and 0.03 ppm, 4.74 and 0.19 ppm, and 2.55 and 0.06 ppm (wet weight),

respectively (Schmitt et al., 1990). Kidwell et al. (1995) conducted an analysis of

all 1984-1985 data from the NCBP study on DDT and its major metabolites (DDE

and DDD) in bottom-feeding and predator fish. The authors reported that there

was no significant difference in residues in these two trophic groups of fish. Mean

tissue concentrations of DDT, DDE, and DDD were 0.03 ± 0.14, 0.21 ± 0.46, and

0.07 ± 0.21 ppm for bottom feeders as compared to 0.03 ± 0.06, 0.24 ± 0.55, and

0.06 ± 0.14 ppm for predator species, respectively. DDE, the only DDT metabolite

surveyed in fish tissue in the EPA National Study of Chemical Residues in Fish,

was detected at more sites than any other single chemical pollutant (99 percent

of the 362 sites sampled) (U.S. EPA, 1992c, 1992d). Maximum, arithmetic mean,

and median concentrations of DDE were 14, 0.295, and 0.058 ppm (wet weight),

respectively. Mean DDE residues from the NSCRF study were highest in bottom

feeders such as carp (0.42 ppm), white sucker (0.08 ppm), and channel catfish

(0.63 ppm) as compared to predator species such as largemouth bass (0.06

ppm), smallmouth bass (0.03 ppb), and walleye (0.03 ppm) (Kuehl et al., 1994).

In 1993, eight states (Alabama, Arizona, California, Delaware, Massachusetts,

Nebraska, New York, and Texas) and the territory of American Samoa had fish

consumption advisories in effect for DDT or its metabolites (RTI, 1993). As of

1998, there were 34 advisories in effect in 11 states and the territory of American

Samoa for DDT and/or one of its metabolites, DDE or DDD (U.S. EPA, 1999c).

In addition, New York has a statewide DDT advisory in effect for mergansers.

Because of the extensive national use of this compound and its widespread

detection in fish tissues, total DDT (i.e., sum of the 4,4'- and 2,4'-homologues of

DDT and of its metabolites, DDE and DDD) should be considered for inclusion in

all state fish and shellfish contaminant monitoring programs. Monitoring sites in

agricultural watersheds should be reviewed to determine the application rate and

acreage where DDT was applied historically. In suburban/urban watersheds, the

degree of historic use of DDT in domestic home and garden applications should

be evaluated. Sites in industrial watersheds should be reviewed to identify

historic sites of DDT production, formulation, or packaging facilities.

4.3.2.3 Dicofol—

Dicofol, one of the three organochlorine target analytes with an active registration,

is a miticide/pesticide that was first registered for use in 1957. Currently, dicofol

is used primarily on cotton, apples, and citrus crops, mostly in California and

Florida (U.S. EPA, 1998c). Dicofol is considered a DDT analog based on its

structure and activity (Hayes and Laws, 1991). In the past, dicofol often contained

9 to 15 percent DDT and its analogs. In 1989, EPA required that these

contaminants constitute less than 0.1 percent of dicofol (HSDB, 1993).
4. TARGET ANALYTES

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Historically, dicofol has been used to control mites on cotton and citrus (60

percent), on apples (10 percent), on ornamental plants and turf (10 percent), and

on a variety of other agricultural products (20 percent) including pears, apricots,

and cherries (Farm Chemical Handbook, 1989), as a seed crop soil treatment, on

vegetables (e.g., beans and corn), and on shade trees (U.S. EPA, 1992c, 1992d).

Dicofol is moderately toxic to laboratory rats and has been assigned to EPA

Toxicity Class III based on an oral LD50 of 587 mg/kg in rats (U.S. EPA, 1998d)

(Appendix F). Technical-grade dicofol induced hepatocellular (liver) carcinomas

in male mice; however, results were negative in female mice and in rats (NCI,

1978) and in a second 2-year feeding study in both sexes of rats (U.S. EPA,

1998d). EPA has classified dicofol as a possible human carcinogen (C)

(Appendix G) (U.S. EPA, 1998c). 

Dicofol was recommended for monitoring by the EPA Office of Water as part of

the Assessment and Control of Bioconcentratable Contaminants in Surface

Waters Program and has been included in two other national monitoring programs

(see Appendix E). Experimental evidence indicates this compound bioaccumulates extensively in bluegill sunfish (BCF from 6,600 to 17,000) (U.S. EPA,

1993a). 

In the EPA National Study of Chemical Residues in Fish, dicofol was detected at

16 percent of the 374 sites monitored (U.S. EPA, 1992c, 1992d). Maximum,

arithmetic mean, and median dicofol concentrations (wet weight basis) were 0.074

ppm, 0.001 ppm, and ND (not detectable). Dicofol concentrations were greater

than the quantification limit (0.0025 ppm) in samples from only 7 percent of the

sites. Most of the sites where dicofol was detected were in agricultural areas

where citrus and other fruits and vegetables are grown (U.S. EPA, 1992c, 1992d).

It should be noted that this national study did not specifically target agricultural

sites where this pesticide historically had been or currently was used. Dicofol

residues in fish could be much higher if sampling were targeted for pesticide

runoff, particularly during the period immediately after field application. Mean

dicofol residues from the NSCRF study were highest in bottom feeders such as

carp (0.88 ppm), white sucker (0.48 ppm), and channel catfish (0.59 ppm) as

compared to predator species such as largemouth bass (0.20 ppm), smallmouth

bass (not detected), and walleye (not detected) (Kuehl et al., 1994). 

In 1993, however, no consumption advisories were in effect for dicofol (RTI,

1993). As of 1998, there were no advisories in effect for this pesticide (U.S. EPA,

1999c). Dicofol should be considered for inclusion in state fish and shellfish

contaminant monitoring programs, in areas where its use is or has been

extensive. States should contact their appropriate state agencies to obtain

information on the historic and current uses of this pesticide. Monitoring sites in

agricultural watersheds should be reviewed to determine the application rate and

acreage where dicofol is currently used and was used historically. Sites in

industrial watersheds should be reviewed to identify historic and current sites of

dicofol production, formulation, or packaging facilities.
4. TARGET ANALYTES

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4.3.2.4 Dieldrin—

Dieldrin is a chlorinated cyclodiene that was widely used in the United States from

1950 to 1974 as a broad spectrum pesticide, primarily on termites and other soildwelling insects and on cotton, corn, and citrus crops. Because the toxicity of this

persistent pesticide posed an imminent danger to human health, EPA banned the

production and most major uses of dieldrin in 1974, and, in 1987, all uses of

dieldrin were voluntarily canceled by industry (see Appendix F).

Dieldrin has been classified by EPA as a probable human carcinogen (B2)

(Appendix G) (IRIS, 1999) and has been identified as a human neurotoxin

(ATSDR, 1991). Dieldrin has been included in seven national monitoring

programs (Appendix E) and has been detected nationwide in freshwater finfish

(Schmitt et al., 1990) and estuarine/marine finfish and shellfish (NOAA, 1987,

1989a). Because it is a metabolite of aldrin, the environmental concentrations of

dieldrin are a cumulative result of the historic use of both aldrin and dieldrin

(Schmitt et al., 1990).

In 1984 and 1985, the U.S. Fish and Wildlife Service collected 321 composite

samples of whole fish from 112 stations nationwide as part of the National

Contaminant Biomonitoring Program. Maximum and geometric mean tissue

concentrations of dieldrin in 1984 were 1.39 and 0.04 ppm (wet weight),

respectively (Schmitt et al., 1990). Kidwell et al. (1995) conducted an analysis of

all 1984-1985 data from the NCBP study on dieldrin in bottom-feeding and

predator fish. These authors reported there was no significant difference in

residues in these two trophic groups of fish. Mean tissue concentrations of dieldrin

were 0.05 ± 0.14 ppm for bottom feeders as compared to 0.04 ± 0.10 ppm for

predator species. Dieldrin was also detected in fish tissue at 60 percent of the

362 sites surveyed as part of the EPA National Survey of Chemical Residues in

Fish (U.S. EPA, 1992c, 1992d). Maximum, arithmetic mean, and median

concentrations of dieldrin in fish tissues were 0.450, 0.028, and 0.004 ppm (wet

weight), respectively. Mean dieldrin residues from the NSCRF study were highest

in bottom feeders such as carp (0.045 ppm), white sucker (0.023 ppm), and

channel catfish (0.015 ppm) as compared to predator species such as largemouth

bass (0.005 ppm), smallmouth bass (0.002 ppm), and walleye (0.002 ppm) (Kuehl

et al., 1994). 

In 1993, three states (Arizona, Illinois, and Nebraska) had issued advisories for

dieldrin contamination in fish (RTI, 1993). As of 1998, there were 23 advisories

in effect in six states (Arizona, California, Colorado, Hawaii, Nebraska, and Texas)

for this pesticide (U.S. EPA, 1999c). Dieldrin should be considered for inclusion

in all state fish and shellfish contaminant monitoring programs in areas where its

use as well as the use of aldrin have been extensive. States should contact their

appropriate state agencies to obtain information on the historic uses of these two

pesticides. Monitoring sites in agricultural watersheds should be reviewed to

determine the application rate and acreage where dieldrin and aldrin were applied

since dieldrin is a degradation product of aldrin. In suburban/urban watersheds,

the degree of historic use of dieldrin and aldrin in domestic home and garden
4. TARGET ANALYTES

4-31

applications should be evaluated. Sites in industrial watersheds should be

reviewed to identify historic sites of dieldrin and aldrin production, formulation, or

packaging facilities.

4.3.2.5 Endosulfan—

Endosulfan is a chlorinated cyclodiene pesticide that is currently in wide use

primarily as a noncontact insecticide for seed and soil treatments (Appendix F).

Two stereohomologues (I and II) exist and exhibit approximately equal

effectiveness and toxicity (Worthing, 1991). 

Endosulfan is highly toxic to laboratory animals and has been assigned to EPA

Toxicity Class I (U.S. EPA, 1998d). To date, no studies have been found

concerning carcinogenicity in humans after oral exposure to endosulfan (ATSDR,

1998c). EPA has classified endosulfan as Group E, evidence of noncarcinogenicity for humans (U.S. EPA, 1999b).

Agricultural runoff is the primary source of this pesticide in aquatic ecosystems.

Endosulfan has been shown to be highly toxic to fish and marine invertebrates

and is readily absorbed in sediments. It therefore represents a potential hazard

in the aquatic environment (Sittig, 1980). However, data are insufficient to assess

nationwide endosulfan contamination (NAS, 1991). Endosulfan has been

included in one national fish contaminant monitoring program—the U.S. EPA

301(h) Program—the (U.S. EPA 301(h) Program—evaluated by the 1993 EPA

Workgroup (Appendix E); however, no information was located related to its

concentrations in fish or shellfish tissue. 

In 1993, no consumption advisories were in effect for endosulfan I or II (RTI,

1993). As of 1998, there were no advisories in effect for this pesticide (U.S. EPA,

1999c). Endosulfan I and II should be considered for inclusion in all state fish and

shellfish contaminant monitoring programs in areas where its use is or has been

extensive. States should contact their appropriate agencies to obtain information

on the historic and current uses of this pesticide. Monitoring sites in agricultural

watersheds should be reviewed to determine the application rate and acreage

where endosulfan currently is used and was used historically. Sites in industrial

watersheds should be reviewed to identify historic and current sites of endosulfan

production, formulation, or packaging facilities.

4.3.2.6 Endrin—

Endrin is a chlorinated cyclodiene that historically was widely used as a broad

spectrum pesticide. Endrin was first registered for use in the United States in

1951. However, recognition of its long-term persistence in soil and its high levels

of mammalian toxicity led to restriction of its use beginning in 1964 and 1979

(U.S. EPA, 1980a; 44 FR 43632) and to final cancellation of its registration in

1984 (U.S. EPA, 1984a) (Appendix F).
4. TARGET ANALYTES

4-32

Endrin is highly toxic to humans (EPA Toxicity Class I) (U.S. EPA, 1998d), with

acute exposures affecting the central nervous system primarily (Sax, 1984). At

present, evidence of both animal and human carcinogenicity of endrin is

considered inadequate, and EPA has classified endrin in Group D, not

classifisable as to human carcinogenicity insufficient information available

(Appendix G) (IRIS, 1999).

Although endrin has been included in five national fish contaminant monitoring

programs (Appendix E), it has not been found widely throughout the United

States. In 1984 and 1985, the U.S. Fish and Wildlife Service collected 321

composite samples of whole fish from 112 stations nationwide as part of the

National Contaminant Biomonitoring Program (Schmitt et al., 1990). Endrin was

detected in freshwater fish at only 29 percent of 112 stations sampled in the

NCBP study. Maximum and geometric mean tissue concentrations of endrin in

1984 were 0.22 and <0.01 ppm (wet weight), respectively (Schmitt et al. 1990).

Endrin was also detected in freshwater and marine species at 11 percent of the

362 sites surveyed in the EPA National Study of Chemical Residues in Fish (U.S.

EPA, 1992c, 1992d). Maximum, arithmetic mean, and median concentrations of

endrin in fish tissues were 0.162 ppm, 0.002 ppm, and not detectable (wet

weight), respectively. Mean endrin residues from the NSCRF study were highest

in bottom feeders such as carp (0.0014 ppm), white sucker (0.0002 ppm), and

channel catfish (0.009 ppm) as compared to predatory species such as

largemouth bass (not detectable), smallmouth bass (not detectable), and walleye

(not detectable) (Kuehl et al., 1994). 

In 1993, no state had issued a fish advisory for endrin (RTI, 1993). As of 1998,

there were no advisories in effect for this pesticide (U.S. EPA, 1999c). Endrin

should be considered for inclusion in all state fish and shellfish contaminant

monitoring programs in areas where its use has been extensive. States should

contact their appropriate agencies to obtain information on the historic uses of

this pesticide. Monitoring sites in agricultural watersheds should be reviewed to

determine the application rate and acreage where endrin was used historically.

Sites in industrial watersheds should be reviewed to identify historic sites of endrin

production, formulation, or packaging facilities.

4.3.2.7 Heptachlor Epoxide—

Heptachlor epoxide is not a formulated pesticide but is a metabolic degradation

product of the pesticides heptachlor and chlordane. It is also found as a

contaminant in heptachlor and chlordane formulations (Appendix F). Heptachlor

epoxide is also more toxic than either parent compound (ATSDR, 1993).

Heptachlor has been used as a persistent, nonsystemic contact and ingested

insecticide on soils (particularly for termite control) and seeds and as a household

insecticide (Worthing, 1991). EPA suspended the major uses of heptachlor in

1978 (ATSDR, 1993). Acute exposures to high doses of heptachlor epoxide in

humans can cause central nervous system effects (e.g., irritability, dizziness,

muscle tremors, and convulsions (U.S. EPA, 1986c). In animals, liver, kidney,

and blood disorders can occur (IRIS, 1999). Exposure to this compound
4. TARGET ANALYTES

4-33

produced an increased incidence of liver carcinomas in rats and mice and

hepatomas in female rats (IRIS, 1999). Heptachlor epoxide has been classified

by EPA as a probable human carcinogen (B2) (Appendix G) (IRIS, 1999).

Heptachlor epoxide has been included in six national fish monitoring programs

(Appendix E) and has been detected widely in freshwater finfish (Schmitt et al.,

1990), but infrequently in bivalves and marine fish (NOAA, 1987, 1989a). In 1984

and 1985, the U.S. Fish and Wildlife Service collected 321 composite samples of

whole fish from 112 stations nationwide as part of the National Contaminant

Biomonitoring Program (Schmitt et al., 1990). Heptachlor epoxide was detected

in freshwater fish at 49 percent of 112 stations sampled in the NCBP study.

Maximum and geometric mean tissue concentrations of heptachlor epoxide in

1984 were 0.29 and 0.01 ppm (wet weight), respectively (Schmitt et al., 1990).

Heptachlor epoxide also was detected in fish tissue at 16 percent of the 362 sites

where it was surveyed in the EPA National Study of Chemical Residues in Fish

(U.S. EPA, 1992c, 1992d). Maximum, arithmetic mean, and median concentrations of heptachlor epoxide were 0.063 ppm, 0.002 ppm, and not detectable

(wet weight). It should be noted that one of the parent compounds, heptachlor

was detected at only 2 percent of the 362 sites where it was surveyed at a

maximum, arithmetic mean, and median concentration of 0.076, 0.0004 ppm, and

not detectable, respectively. The five degradation products of chlordane were

detected at from 27 to 77 percent of these same sites (see Section 4.3.2.1 for a

discussion of chlordane). Mean heptachlor epoxide residues from the NSCRF

study were highest in bottom feeders such as carp (0.004 ppm), white sucker

(0.001 ppm), and channel catfish (0.0005 ppm) as compared to predator species

such as largemouth bass (0.0003 ppm), smallmouth bass (0.00007 ppm), and

walleye (0.0002 ppm) (Kuehl et al., 1994). 

In 1993, only Nebraska had fish advisories for heptachlor epoxide contamination

(RTI, 1993). As of 1998, there was only one advisory in effect, in Texas, for this

pesticide degradation product (U.S. EPA, 1999c). Heptachlor epoxide should be

considered for inclusion in all state fish and shellfish monitoring programs in areas

where the use of heptachlor or chlordane have been extensive. States should

contact their appropriate agencies to obtain information on the historic uses of

these pesticides. Monitoring sites in agricultural watersheds should be reviewed

to determine the application rate and acreage where heptachlor and chlordane

were historically used since both of these pesticides degrade to heptachlor

epoxide. In suburban/urban watersheds, the degree of historic use of heptachlor

and chlordane in domestic home and garden applications should be evaluated.

Sites in industrial watersheds also should be reviewed to identify historic sites of

heptachlor and chlordane production, formulation, or packaging facilities.

4.3.2.8 Hexachlorobenzene—

Hexachlorobenzene is a fungicide that was widely used as a seed protectant in

the United States until 1984 (Appendix F). The use of hexachlorobenzene and the

presence of hexachlorobenzene residues in food are banned in many countries
4. TARGET ANALYTES

4-34

including the United States (Worthing, 1991). Registration of hexachlorobenzene

as a pesticide was voluntarily canceled in 1984 (Morris and Cabral, 1986).

The toxicity of this compound is minimal; it has been given an EPA toxicity

classification of IV (i.e., oral LD50 greater than 5,000 ppm in laboratory animals

(U.S. EPA, 1998d). However, nursing infants are particularly susceptible to

hexachlorobenzene poisoning as lactational transfer can increase infant tissue

levels to two to five times maternal tissue levels (ATSDR, 1996).

Hexachlorobenzene is a known animal carcinogen (ATSDR, 1996) and has been

classified by EPA as a probable human carcinogen (B2) (Appendix G) (IRIS,

1999).

Of the chlorinated benzenes, hexachlorobenzene is the most widely monitored

(Worthing, 1991). It was included as a target analyte in seven of the major

monitoring programs reviewed by the 1993 Workgroup (Appendix E). In 1984 and

1985, the U.S. Fish and Wildlife Service collected 321 composite samples of

whole fish from 112 stations nationwide as part of the National Contaminant

Biomonitoring Program (Schmitt et al., 1990). Hexachlorobenzene was detected

in freshwater fish at 19 percent of 112 stations sampled in the NCBP study.

Maximum and geometric mean tissue concentrations of hexachlorobenzene in

1984 were 0.41 and <0.01 ppm (wet weight), respectively (Schmitt et al., 1990).

Kidwell et al. (1995) conducted an analysis of all 1984-1985 data from the NCBP

on hexachlorobenzene in bottom-feeding and predator fish. The authors reported

that there was no significant difference in residues in these two trophic groups.

Mean tissue concentrations of HCB were 0.00 ± 0.01 and 0.01 ± 0.04 ppm,

respectively, for bottom feeders and predator species. Hexachlorobenzene also

was detected in fish tissue at 46 percent of the 362 sites where it was surveyed

in the EPA National Study of Chemical Residues in Fish (U.S. EPA, 1992c,

1992d). Maximum, arithmetic mean, and median concentrations were 0.913 ppm,

0.006 ppm, and not detectable (wet weight), respectively. Mean hexachlorobenzene residues from the NSCRF study were highest in bottom feeders such as

carp (0.0036 ppm), white sucker (0.0036 ppm), and channel catfish (0.0024 ppm)

as compared to predator species such as largemouth bass (0.0002 ppm),

smallmouth bass (0.0004 ppm), and walleye (0.0001 ppm) (Kuehl et al., 1994).

In 1993, Louisiana and Ohio had issued advisories for hexachlorobenzene

contamination in fish and shellfish (RTI, 1993). As of 1988, there were three

advisories in effect in these two states for this pesticide (U.S. EPA, 1999c).

Hexachlorobenzene should be considered for inclusion in all state fish and

shellfish monitoring programs. Monitoring sites in agricultural watersheds should

be reviewed to determine the application rate and acreage where hexachlorobenzene was historically used. Sites in industrial watersheds also should be

reviewed to identify historic sites of hexachlorobenzene as well as other organochlorine pesticide production, formulation, or packaging facilities since hexachlorobenzene was used as an intermediate in the chemical synthesis of many organochlorine pesticides.
4. TARGET ANALYTES

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4.3.2.9 Lindane—

Lindane is a mixture of homologues of hexachlorocyclohexane (C6H6Cl6), whose

major component (99 percent) is the gamma isomer. It is commonly referred to

as either -HCH (hexachlorocyclohexane) or -BHC (benzene hexachloride).

Lindane is used primarily in seed treatments, soil treatments for tobacco

transplants, foliage applications on fruit and nut trees and vegetables, and wood

and timber protection. Lindane is used as a therapeutic scabicide, pediculicide,

and ectoparasiticide for humans and animals (Merck Index 1989). Since 1985,

many uses of lindane have been banned or restricted (see Appendix F) and its

application is permitted only under supervision of a certified applicator (U.S. EPA,

1985c). In 1993, EPA issued a “Notice of Receipt of a Request for Amendments

to Delete Uses” for several formulations of lindane provider, 99.5 percent

technical, and dust concentrate, which would delete from the pesticide label most

uses of lindane for agricultural crops and use on animals and humans (EPA

1993).

Lindane is a neurotoxin (assigned to EPA Toxicity Class II) (U.S. EPA, 1998d)

and has been found to cause aplastic anemia in humans (Worthing, 1991).

Lindane has been classified by EPA as a probable/possible human carcinogen

(B2/C) (Appendix G) (U.S. EPA, 1999b).

Lindane has been included in seven major fish contaminant monitoring programs

(Appendix E). This pesticide has been detected in freshwater fish (Schmitt et al.,

1990) and in marine fish and bivalves (NOAA, 1987, 1989a) nationwide. In 1984

and 1985, the U.S. Fish and Wildlife Service collected 321 composite samples of

whole fish from 112 stations nationwide as part of the National Contaminant

Biomonitoring Program (Schmitt et al., 1990). Lindane was detected in freshwater

fish at 47 percent of 112 stations sampled in the NCBP study. Maximum and

geometric mean tissue concentrations of lindane in 1984 were 0.40 and <0.01

ppm (wet weight), respectively (Schmitt et al., 1990). Kidwell et al. (1995)

conducted an analysis of all 1984-1985 data from the NCBP study on lindane in

bottom-feeding and predator fish. These authors reported there was no

significant difference in residues in these two trophic groups of fish. Lindane also

was detected in fish tissue at 42 percent of 362 sites surveyed in the EPA

National Study of Chemical Residues in Fish (U.S. EPA, 1992c, 1992d).

Maximum, arithmetic mean, and median lindane concentrations were 0.083 ppm,

0.003 ppm, and not detectable (wet weight), respectively. Mean lindane residues

from the NSCRF study were highest in bottom feeders such as carp (0.0043

ppm), white sucker (0.0017 ppm), and channel catfish (0.0032 ppm) as compared

to predator species such as largemouth bass (0.00007 ppm), smallmouth bass

(0.00015 ppm), and walleye (not detectable) (Kuehl et al., 1994). 

In 1993, although it had been widely monitored and widely detected, no

consumption advisories were in effect for lindane (RTI, 1993). As of 1998, there

were no advisories in effect for this pesticide (U.S. EPA, 1999c). Lindane should

be considered for inclusion in all state fish and shellfish monitoring programs in

areas where its use has been extensive. States should contact their appropriate
4. TARGET ANALYTES

4-36

agencies to obtain information on the historic and current uses of this pesticide.

Monitoring sites in agricultural watersheds should be reviewed to determine the

application rate and acreage where lindane was used historically. In suburban/

urban watersheds, the degree of historic use of lindane in domestic home and

garden applications should be evaluated. Sites in industrial watersheds should

be reviewed to identify historic and current sites of lindane production,

formulation, or packaging facilities.

4.3.2.10 Mirex—

Mirex is a chlorinated cyclodiene pesticide that was used in large quantities in the

United States from 1962 through 1975 primarily for control of fire ants in the

Southeast and Gulf Coast states and, more widely, under the name Dechlorane

as a fire retardant and polymerizing agent in plastics (Kaiser, 1978; Kutz et al.,

1985) (Appendix F).

Mirex has been assigned to EPA Toxicity Class II on the basis of an oral LD50 in

rats of 368 mg/kg (ATSDR, 1995; U.S. EPA, 1998d) (Appendix F). Mirex has

been assigned a carcinogenicity classification of group B2, probable human

carcinogen (HEAST, 1997). EPA instituted restrictions on the use of mirex in

1975, and, thereafter, the U.S. Department of Agriculture (USDA) suspended the

fire ant control program (Hodges, 1977).

Mirex has been included in seven major fish contaminant monitoring programs

(Appendix E). It has been found primarily in the Southeast, Gulf Coast, and the

Great Lakes regions (Kutz et al., 1985; NAS, 1991; Schmitt et al., 1990). In 1984

and 1985, the U.S. Fish and Wildlife Service collected 321 composite samples of

whole fish from 112 stations nationwide as part of the National Contaminant

Biomonitoring Program (NCBP) (Schmitt et al., 1990). Mirex was detected in

freshwater fish at 13 percent of 112 stations sampled in the NCBP study.

Maximum and geometric mean tissue concentrations of mirex in 1984 were 0.44

and <0.01 ppm (wet weight), respectively (Schmitt et al., 1990). Kidwell et al.

(1995) conducted an analysis of all 1984-1985 data from the NCBP study on

mirex in bottom-feeding and predator fish. These authors reported there was no

significant difference in residues in these two trophic groups of fish. Mean tissue

concentrations of mirex were 0.00 ± 0.04 and 0.01 + 0.05 ppm, respectively, for

bottom feeders and predator species. Mirex also was detected in fish tissue at 38

percent of 362 sites surveyed in the EPA National Study of Chemical Residues

in Fish (NSCRF) (U.S. EPA, 1992c, 1992d). Maximum, arithmetic mean, and

median mirex concentrations were 0.225 ppm, 0.004 ppm, and not detectable

(wet weight), respectively. Mean mirex residues from the EPA NSCRF study

were highest in bottom feeders such as carp (0.0037 ppm), white sucker (0.0044

ppm), and channel catfish (0.0146 ppm) as compared to predator species such

as largemouth bass (0.0002 ppm), smallmouth bass (0.002 ppm), and walleye

(0.00008 ppm) (Kuehl et al., 1994). 

In 1993, three states (New York, Ohio, and Pennsylvania) had issued fish

advisories for mirex (RTI, 1993). As of 1998, there were 11 advisories in effect
4. TARGET ANALYTES

4-37

in these same three states for this pesticide (U.S. EPA, 1999c). New York has

a statewide advisory in effect for mergansers. Mirex should be considered for

inclusion in all state fish and shellfish monitoring programs in areas where its use

has been extensive. States should contact their appropriate agencies to obtain

information on the historic uses of this pesticide. Monitoring sites in agricultural

watersheds should be reviewed to determine the application rate and acreage

where mirex was used historically. In suburban/urban watersheds, the degree of

historic use of mirex in domestic home and garden applications should be

evaluated. Sites in industrial watersheds should be reviewed to identify historic

sites of mirex production, formulation, or packaging facilities.

4.3.2.11 Toxaphene—

Toxaphene is an organochlorine pesticide composed of a complex mixture of

chlorinated camphenes (chlorinated bornanes and some bornenes) that was first

registered for use in the United States in 1947. It was commercially produced by

the chlorination of camphenes derived from pine trees. It has been estimated that

the commercial mixture of toxaphene contained at least 670 congeners with the

majority of these having 6 to 10 chlorines (Jansson and Wideqvist, 1983).

Historically, this compound was used in the United States as an insecticide

primarily on cotton (Hodges, 1977). In addition, toxaphene was used as a

piscicide for rough fish in the 1950s and 1960s in North America and was the

replacement for DDT after DDT’s use was severely restricted in 1972 (Saleh,

1991). Partly as a consequence of the ban on the use of DDT imposed in 1972,

toxaphene was for many years the most heavily used pesticide in the United

States (Eichers et al., 1978). In 1982, toxaphene's registration for most uses was

canceled (47 FR 53784) and all uses were banned in 1990 (55 FR 31164-31174).

Toxaphene is a global pollutant whose chemical-physical properties make it a

candidate for long-range atmospheric transport via the cold condensation effect

once it is released into the environment (Wania and Mackay, 1993, 1996).

Like many of the other organochlorine pesticides, toxaphene has been assigned

to EPA Toxicity Class II (U.S. EPA, 1998d) (Appendix F). Some components of

toxaphene may accumulate in body fat. Toxaphene has been classified by EPA

as a probable human carcinogen (B2) (Appendix G) (IRIS, 1999).

Toxaphene has been included in four major fish contaminant monitoring programs

(Appendix E). It has been detected frequently in both freshwater fish (Schmitt et

al., 1990) and estuarine species (NOAA, 1989a) but is only consistently found in

Georgia, Texas, and California (NAS, 1991). In 1984 and 1985, the U.S. Fish and

Wildlife Service collected 321 composite samples of whole fish from 112 stations

nationwide as part of the National Contaminant Biomonitoring Program (Schmitt

et al., 1990). Toxaphene was detected in freshwater fish at 69 percent of 112

stations sampled in the NCBP study. Maximum and geometric mean tissue

concentrations of toxaphene in 1984 were 8.2 and 0.14 ppm (wet weight),

respectively (Schmitt et al., 1990). Kidwell et al. (1995) conducted an analysis of

all 1984-1985 data from the NCBP study on toxaphene in bottom-feeding and

predatory fish species. These authors reported there was no significant difference
4. TARGET ANALYTES

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in residues in these two trophic groups of fish. Mean tissue concentrations of

toxaphene were 0.19 ± 0.63 and 0.17 + 0.35 ppm, respectively, for bottom

feeders and predator species. 

In 1993, two states (Arizona and Texas) had fish advisories in effect for

toxaphene (RTI, 1993). As of 1988, there were six advisories in effect in four

states (Arizona, Georgia, Oklahoma, and Texas) for this pesticide (U.S. EPA,

1999c). Toxaphene should be considered for inclusion in all state fish and

shellfish monitoring programs in areas where its use has been extensive. States

should contact their appropriate agencies to obtain information on the historic

uses of this pesticide. Monitoring sites in agricultural watersheds should be

reviewed to determine the application rate and acreage where toxaphene was

used historically. Sites in industrial watersheds should be reviewed to identify

historic sites of toxaphene production, formulation, or packaging facilities.

4.3.3 Organophosphate Pesticides

The following organophosphate pesticides are recommended as target analytes

in screening studies: chlorpyrifos, diazinon, disulfoton, ethion, and terbufos

(Appendix E). These pesticides share two distinct features that differentiate them

from the organochlorines. Organophosphate pesticides are generally more

acutely toxic to vertebrates than organochlorine pesticides and exert their toxic

action by inhibiting the activity of cholinesterase (ChE), one of the vital nervous

system enzymes. In addition, organophosphates are chemically unstable (they

are all slowly hydrolyzed by water) and thus are less persistent in the

environment. It is this latter feature that made them attractive alternatives to the

organochlorine pesticides that were used extensively in agriculture from the 1940s

to the early 1970s. 

With the exception of chlorpyrifos, none of the organophosphates has been

included in any of the national fish contaminant monitoring programs evaluated

by the EPA 1993 Workgroup and none of these pesticides (including chlorpyrifos)

has triggered state fish consumption advisories. All of the organophosphate

pesticides have active pesticide registrations and have been recommended for

monitoring because they have an EPA Toxicity Classification of I or II

(Appendix F), BCFs >300, and a half-life of 30 days or more in the environment

and their use profiles suggest they could be potential problems in some

agricultural watersheds.

The target organophosphates are used in agriculture throughout the United

States, particularly in areas under intensive cultivation (row crops, orchards, fruits,

and vegetables). Bioconcentration studies indicate they can accumulate in fish

and, because they are known human neurotoxins, the potential exists for human

health effects from consuming contaminated fish. For this reason, federal

regulations are in effect that set maximum application rates and minimize use

near waterbodies. At the time of this writing, no fish consumption advisories for

these target analytes have yet been issued; however, state agencies should be

aware of special circumstances that could result in their accumulation in fish. In
4. TARGET ANALYTES

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addition to chemical spills and misapplications, heavy and repeated rainfall shortly

after application may wash pesticides off of plants and into streams. Signs of

acute organophosphate pollution may include erratic swimming behavior in fish

or fish kills.

States should contact their appropriate agencies to obtain information on both the

historic and current uses of these pesticides. With the exception of ethion, which

is used almost exclusively on citrus, the target organophosphates are used on a

wide variety of crops. In addition, chlorpyrifos and diazinon have significant uses

in domestic and commercial pest control in suburban/urban areas (Robinson et

al., 1994). If a state determines that high concentrations of these pesticides may

be present in its agricultural watersheds, sampling should be conducted during

late spring or early summer within 1 to 2 months following pesticide application to

maximize detection of these compounds in fish tissues. In general, the

organophosphates are degraded relatively rapidly in the environment and

metabolized relatively rapidly by fish, so timing of the sampling program is a more

important consideration for this class of pesticides. Additional discussion of

appropriate sampling times for fish contaminant monitoring programs is provided

in Section 6.1.1.5.

All of the target organophosphates are members of the organothiophosphate

group of insecticides. They are all metabolized in the liver to their active form,

referred to as an “oxon” (e.g., chlorpyrifos is activated to chlorpyrifos oxon)

(Klaasen, 1996). The oxons are approximately 300- to 1,000-fold more toxic than

the parent compounds; however, they are also less lipid-soluble than the parent

compounds and, therefore, are expected to be less likely to bioaccumulate in fish

tissue. In another laboratory study where chlorpyrifos was fed to channel catfish,

only chlorpyrifos and its inactive metabolites were found; the oxon was not

detected in any tissue (Barron et al., 1991). No information is available on the

presence of the oxon metabolites in fish tissue for the other organophosphates.

Note: The potential human toxicity of the organophosphates is undergoing

reassessment by EPA at this time as a result of the provisions of the Food Quality

Protection Act of 1996. For more information, consult the EPA Office of Pesticide

Programs webpage available on the Internet at: 

http://www.epa.gov.pesticides/op.

4.3.3.1 Chlorpyrifos—

This organophosphate pesticide was first introduced in 1965 to replace the more

persistent organochlorine pesticides (e.g., DDT) (U.S. EPA, 1986c) and has been

used for a broad range of insecticide applications (Appendix F). Chlorpyrifos is

used primarily to control soil and foliar insects on cotton, peanuts, and sorghum

(Worthing, 1991; U.S. EPA, 1986c). Chlorpyrifos is also used to control rootinfesting and boring insects on a variety of fruits (e.g., apples, bananas, citrus,

grapes), nuts (e.g., almonds, walnuts), vegetables (e.g., beans, broccoli, brussel

sprouts, cabbage, cauliflower, peas, and soybeans), and field crops (e.g., alfalfa

and corn) (U.S. EPA, 1984c). As a household insecticide, chlorpyrifos has been
4. TARGET ANALYTES

4-40

used to control ants, cockroaches, fleas, and mosquitoes (Worthing, 1991) and

is registered for use in controlling subsurface termites in California (U.S. EPA,

1983a). Based on use application, 48 percent of chlorpyrifos use is agricultural

and 52 percent is nonagricultural (U.S. EPA, 2000b). Chlorpyrifos is also used

by the general public for home, lawn, and garden insect control (ATSDR, 1997).

Note: As a result of the reassessment conducted under the Food Quality Act of

1996, use patterns of chlorpyrifos will change significantly by the end of 2001. In

particular, virtually all indoor and outdoor residential use will end, as well as all

agricultural use on tomatoes. Agricultural use of chlorpyrifos on apples and

grapes will be reduced substantially (U.S. EPA, 2000b).

Chlorpyrifos has a moderate mammalian toxicity and has been assigned to EPA

Toxicity Class II based on oral feeding studies (U.S. EPA, 1998d). No

carcinogenicity was found in chronic feeding studies with rats, mice, and dogs

(U.S. EPA, 1983a). Because chlorpyrifos did not increase the incidence of

cancer in feeding studies on rats and mice (U.S. EPA, 1999b, U.S. EPA, 2000b)

EPA has classified chlorpyrifos in Group E (Appendix G) (U.S. EPA, 2000b).

Experimental evidence indicates this compound bioaccumulates in rainbow trout

(BCF from 1,280 to 3,903) (U.S. EPA, 1993a). 

Chlorpyrifos has been included in one national monitoring program reviewed by

the EPA 1993 Workgroup, the EPA National Study of Chemical Residues in Fish

(NSCRF) (see Appendix E). In this study, chlorpyrifos was detected at 26 percent

of sites sampled nationally (U.S. EPA, 1992c, 1992d). Eighteen percent of the

sites with relatively high concentrations (0.0025 to 0.344 ppm) were scattered

throughout the East, Midwest, and in California; the highest mean concentrations

detected (0.060 to 0.344 ppm) were found either in agricultural areas or in urban

areas with a variety of nearby industrial sources. Maximum, arithmetic mean, and

median tissue concentrations (wet weight) of chlorpyrifos were 0.344 ppm, 0.004

ppm, and not detectable, respectively. Mean chlorpyrifos residues from the

NSCRF study were highest in bottom feeders such as carp (0.0082 ppm), white

sucker (0.0018 ppm), and channel catfish (0.007 ppm) as compared to predator

species such as largemouth bass (0.00028 ppm), smallmouth bass (0.00008

ppm), and walleye (0.00004 ppm) (Kuehl et al., 1994). It should be noted that this

national study did not specifically target agricultural sites where this pesticide

historically had been used or is currently used. Chlorpyrifos residues in fish could

be much higher if sampling were targeted for pesticide runoff, especially during

the period immediately after field application. 

In 1993, no consumption advisories were in effect for chlorpyrifos (RTI, 1993).

As of 1998, there were no advisories in effect for this pesticide (U.S. EPA, 1999c).

Chlorpyrifos should be considered for inclusion in state fish and shellfish

contaminant monitoring programs in areas where its use is or has been extensive.

States should contact their appropriate agencies to obtain information on the

historic and current uses of this pesticide. Monitoring sites in agricultural watersheds should be reviewed to determine the application rate and acreage where

chlorpyrifos is currently used or was used historically. In suburban/urban water-
4. TARGET ANALYTES

4-41

sheds, the degree of historic and current use of chlorpyrifos in domestic home

and garden applications should be evaluated. Sites in industrial watersheds also

should be reviewed to identify historic and current sites of chlorpyrifos production,

formulation, or packaging facilities.

4.3.3.2 Diazinon—

Diazinon is a phosphorothiate insecticide and nematicide that was first registered

in 1952 for control of soil insects and pests of fruits, vegetables, tobacco, forage,

field crops, range, pasture, grasslands, and ornamentals; for control of

cockroaches and other household insects; for control of grubs and nematodes in

turf; as a seed treatment; and for fly control (U.S. EPA, 1986d). Diazinon is also

used by the general public for home, lawn, and garden insect control (Appendix F)

(ATSDR, 1996).

Diazinon is moderately toxic to mammals and has been assigned to EPA Toxicity

Class II based on oral toxicity tests (U.S. EPA, 1998d) (Appendix F). Diazinon

was not found to be carcinogenic in rats and mice (ATSDR, 1996). Because of

inadequate evidence of carcinogenicity, EPA has classified diazinon as “not likely

to be a human carcinogen”) (Appendix G) (U.S. EPA, 1998d). This compound is

also highly toxic to birds, fish, and other aquatic invertebrates (U.S. EPA, 1986d).

Diazinon was not included in any national fish contaminant monitoring program

evaluated by the EPA 1993 Workgroup (Appendix E). Experimental evidence

indicates this compound accumulates in trout (BCF of 542) (U.S. EPA, 1993a).

In 1993, no consumption advisories were in effect for diazinon (RTI, 1993). As of

1998, there were no advisories in effect for this pesticide (U.S. EPA, 1999c).

Diazinon should be considered for inclusion in state fish and shellfish contaminant

monitoring programs in areas where its use is or has been extensive. States

should contact their appropriate agencies to obtain information on the historic and

current uses of this pesticide. Monitoring sites in agricultural watersheds should

be reviewed to determine the application rate and acreage where diazinon is

currently used or was used historically. In suburban/urban watersheds, the

degree of historic and current use of diazinon in domestic home and garden

applications should be evaluated. Sites in industrial watersheds should be

reviewed to identify historic and current sites of diazinon production, formulation,

or packaging facilities.

4.3.3.3 Disulfoton—

Disulfoton is a multipurpose systemic insecticide and acaricide first registered in

1958 for use as a side dressing, broadcast, or foliar spray in the seed furrow to

control many insect and mite species and as a seed treatment for sucking insects

(Appendix F) (Farm Chemicals Handbook, 1989).

Disulfoton is highly toxic to all mammalian systems and has been assigned to

EPA Toxicity Class I on the basis of all routes of exposure (U.S. EPA, 1998d).
4. TARGET ANALYTES

4-42

Disulfoton was not found to be carcinogenic in dogs, rats, or mice (ATSDR, 1995).

Because of inadequate evidence of carcinogenicity, EPA has classified disulfoton

as Group E, evidence of noncarcinogenicity for humans (Appendix G) (U.S. EPA,

1999b).

Disulfoton was not included in any national fish contaminant monitoring program

evaluated by the EPA 1993 Workgroup (Appendix E). Experimental evidence

indicates this compound accumulates in fish (BCF from 460 to 700) (U.S. EPA,

1993a). 

In 1993, no consumption advisories were in effect for disulfoton (RTI, 1993). As

of 1998, there were no advisories in effect for this pesticide (U.S. EPA, 1999c).

Disulfoton should be considered for inclusion in state fish and shellfish

contaminant monitoring programs in areas where its use is or has been extensive.

States should contact their appropriate agencies to obtain information on the

historic and current uses of this pesticide. Monitoring sites in agricultural watersheds should be reviewed to determine the application rate and acreage where

disulfoton currently is used or was used historically. Sites in industrial watersheds

also should be reviewed to identify historic and current sites of disulfoton production, formulation, or packaging facilities.

4.3.3.4 Ethion—

Ethion is a multipurpose insecticide and acaricide that has been registered since

1965 for use on a wide variety of nonfood crops (turf, evergreen plantings, and

ornamentals), food crops (seed, fruit, nut, fiber, grain, forage, and vegetables),

and for domestic outdoor uses around dwellings and for lawns (Appendix F)

(Farm Chemicals Handbook, 1989). Application to citrus crops accounts for 86

to 89 percent of the ethion used in the United States. The remaining 11 to

14 percent is applied to cotton and a variety of fruit and nut trees and vegetables.

Approximately 55 to 70 percent of all domestically produced citrus fruits are

treated with ethion (U.S. EPA, 1989e).

Acute oral toxicity studies have shown that technical-grade ethion is moderately

toxic to mammals (EPA Toxicity Class II) (U.S. EPA, 1998d). Ethion was not

found to be carcinogenic in rats and mice (U.S. EPA, 1989e). EPA has classified

ethion in Group E–evidence of noncarcinogenicity for humans (Appendix G) (U.S.

EPA, 1999b). 

Ethion was not included in any national fish contaminant monitoring program

evaluated by the EPA 1993 Workgroup (Appendix E). Experimental evidence

indicates this compound accumulates in bluegill sunfish (BCF from 880 to 2,400)

(U.S. EPA, 1993a).

In 1993, no consumption advisories were in effect for ethion (RTI, 1993). As of

1998, there were no advisories in effect for this pesticide (U.S. EPA, 1999c).

Ethion should be considered for inclusion in state fish and shellfish contaminant

monitoring programs in areas where its use is or has been extensive. States
4. TARGET ANALYTES

4-43

should contact their appropriate agencies to obtain information on the historic and

current uses of this pesticide. Monitoring sites in agricultural watersheds should

be reviewed to determine the application rate and acreage where ethion currently

is used or was used historically. In suburban/urban watersheds, the degree of

historic and current use of ethion in domestic home and garden applications

should be evaluated. Sites in industrial watersheds also should be reviewed to

identify historic and current sites of ethion production, formulation, or packaging

facilities.

4.3.3.5 Terbufos—

Terbufos is a systemic organophosphate insecticide and nematicide registered in

1974 principally for use on corn, sugar beets, and grain sorghum. The primary

method of application involves direct soil incorporation of a granular formulation

(Farm Chemicals Handbook, 1989). Two soil metabolites of terbufos, terbufos

sulfoxide and terbufos sulfone, are also toxic to humans and are found at sites

where terbufos has been applied (U.S. EPA, 1995)

Terbufos is highly toxic to humans and has been assigned to EPA Toxicity Class I

(U.S. EPA, 1998d) (Appendix F). Terbufos was not found to be carcinogenic in

rats and mice (U.S. EPA, 1995j). EPA has assigned terbufos to carcinogenicity

classification E, evidence of noncarcinogenicity for humans (U.S. EPA, 1998d)

(Appendix G). Terbufos is also highly toxic to birds, fish, and other aquatic

invertebrates (U.S. EPA, 1985d).

Terbufos was not included in any national fish contaminant monitoring program

evaluated by the EPA 1993 Workgroup (Appendix E). Experimental evidence

indicates this compound accumulates in fish (BCF from 320 to 1,400) (U.S. EPA,

1993a). 

In 1993, no consumption advisories were in effect for terbufos (RTI, 1993). As of

1998, there were no advisories in effect for this pesticide (U.S. EPA, 1999c).

Terbufos and its toxic metabolites should be considered for inclusion in state fish

and shellfish contaminant monitoring programs in areas where its use is or has

been extensive. States should contact their appropriate agencies to obtain

information on the historic and current uses of this pesticide. Monitoring sites in

agricultural watersheds should be reviewed to determine the application rate and

acreage where terbufos currently is used or was used historically. Sites in industrial watersheds also should be reviewed to identify historic and current sites of

terbufos production, formulation, or packaging facilities.

4.3.4 Chlorophenoxy Herbicides

Chlorophenoxy herbicides, which include oxyfluorfen, are nonselective foliar

herbicides that are most effective in hot weather (Ware, 1978).
4. TARGET ANALYTES

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4.3.4.1 Oxyfluorfen—

Oxyfluorfen is a pre- and postemergence herbicide with an active registration that

has been registered since 1979 for use to control a wide spectrum of annual

broadleaf weeds and grasses in apples, artichokes, corn, cotton, jojoba, tree

fruits, grapes, nuts, soybeans, spearmint, peppermint, and certain tropical

plantation and ornamental crops (Appendix F) (Farm Chemicals Handbook,

1989).

Oxyfluorfen is of low toxicity to mammals (oral LD50 in rats >5,000 mg/kg) and has

been assigned to EPA Toxicity Class IV (U.S. EPA, 1998d) (Hayes and Lawes,

1991). There is also evidence of carcinogenicity (liver tumors) in mice (U.S. EPA,

1993a) and therefore oxyfluorfen has been classified by EPA as a possible human

carcinogen (C) (Appendix G) (U.S. EPA, 1999b).

Oxyfluorfen was not included in any national fish contaminant monitoring program

evaluated by the EPA 1993 Workgroup (Appendix E). Experimental evidence

indicates this herbicide accumulates in bluegill sunfish (BCF from 640 to 1,800)

(U.S. EPA, 1993a).

In 1993, no consumption advisories were in effect for oxyfluorfen (RTI, 1993). As

of 1998, there were no advisories in effect for this herbicide (U.S. EPA, 1999c).

Oxyfluorfen should be considered for inclusion in state fish and shellfish

contaminant monitoring programs in areas where its use is or has been extensive.

States should contact their appropriate agencies to obtain information on the

historic and current uses of this pesticide. Monitoring sites in agricultural watersheds should be reviewed to determine the application rate and acreage where

oxyfluorfen currently is used or was used historically. Sites in industrial watersheds also should be reviewed to identify historic and current sites of oxyfluorfen

production, formulation, or packaging facilities.

4.3.5 Polycyclic Aromatic Hydrocarbons

PAHs are base/neutral organic compounds that have a fused ring structure of two

or more benzene rings. PAHs are also commonly referred to as polynuclear

aromatic hydrocarbons (PNAs). PAHs with two to five benzene rings (i.e., 10 to

24 skeletal carbons) are generally of greatest concern for environmental and

human health effects (Benkert, 1992). These PAHs have been identified as the

most important with regard to human exposure (ATSDR, 1995):

• Acenaphthene

• Acenaphthylene

• Anthracene

• Benz[a]anthracene

• Benzo[a]pyrene

• Benzo[e]pyrene

• Benzo[b]fluoranthene

• Benzo[k]fluoranthene

• Benzo[j]fluoranthene

• Benzo[g,h,i]perylene

• Chrysene

• Dibenz[a,h]anthracene

• Fluoranthene

• Fluorene

• Indeno[1,2,3-cd]pyrene

• Phenanthrene
4. TARGET ANALYTES

4-45

• Pyrene.

The metabolites of many of the high-molecular-weight PAHs (e.g., benz[a] anthracene, benzo[a]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, chrysene,

dibenz[a,h]anthracene, indeno[1,2,3-cd]pyrene, and benzo[g,h,i]perylene) have

been shown in laboratory test systems to be carcinogens, cocarcinogens,

teratogens, and/or mutagens (Moore and Ramamoorthy, 1984; ATSDR 1995).

Benzo[a]pyrene, one of the most widely occurring and potent PAHs, and six other

PAHs (e.g., benz[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene,

chrysene, dibenz[a,h]anthracene, indeno[1,2,3-cd]pyrene) have been classified

by EPA as probable human carcinogens (B2) (IRIS, 1999). Evidence for the

carcinogenicity of PAHs in humans comes primarily from epidemiologic studies

that have shown an increased mortality due to lung cancer in humans exposed

to PAH-containing coke oven emissions, roof-tar emissions, and cigarette smoke

(ATSDR, 1995).

PAHs are ubiquitous in the environment and usually occur as complex mixtures

with other toxic chemicals. They are components of crude and refined petroleum

products and of coal. They are also produced by the incomplete combustion of

organic materials. Many domestic and industrial activities involve pyrosynthesis

of PAHs, which may be released into the environment in airborne particulates or

in solid (ash) or liquid byproducts of the pyrolytic process. Domestic activities that

produce PAHs include cigarette smoking, home heating with wood or fossil fuels,

waste incineration, broiling and smoking foods, and use of internal combustion

engines. Industrial activities that produce PAHs include wood preserving, coal

coking; production of carbon blacks, creosote, and coal tar; petroleum refining;

synfuel production from coal; and use of Soderberg electrodes in aluminum

smelters and ferrosilicum and iron works (ATSDR, 1995; Neff, 1985). Historic

coal gasification sites have also been identified as significant sources of PAH

contamination (ATSDR, 1995).

Major sources of PAHs found in marine and fresh waters include biosynthesis

(restricted to anoxic sediments), spillage and seepage of fossil fuels, discharge

of domestic and industrial wastes, atmospheric deposition, and runoff (Neff,

1985). Urban stormwater runoff contains PAHs from leaching of asphalt roads,

wearing of tires, deposition from automobile exhaust, and oiling of roadsides and

unpaved roadways with crankcase oil (ATSDR, 1995; MacKenzie and Hunter,

1979). Solid PAH-containing residues from activated sludge treatment facilities

have been disposed of in landfills or in the ocean (ocean dumping was banned in

1989). Although liquid domestic sewage contains <1 μg/L total PAH, the total

PAH content of industrial sewage is 5 to 15 μg/L (Borneff and Kunte, 1965) and

that of sewage sludge is 1 to 30 mg/kg (Grimmer et al., 1978; Nicholls et al.,

1979).

In most cases, there is a direct relationship between PAH concentrations in river

water and the degree of industrialization and human activity in the surrounding

watersheds. Rivers flowing through heavily industrialized areas may contain 1 to
4. TARGET ANALYTES

4-46

5 ppb total PAH, compared to unpolluted river water, ground water, or seawater

that usually contains less than 0.1 ppb PAH (Neff, 1979).

PAHs can accumulate in aquatic organisms from water, sediments, and food.

BCFs of PAHs in fish, crustaceans, and bivalves have frequently been reported

to be in the range of 12 to 9,200 for fish, 200 to 134,248 for crustaceans, and 8

to 242 for bivalves based on short-term exposure studies typically less than

7 days duration (Eisler, 1987). In general, bioconcentration was greater for the

higher molecular weight PAHs than for the lower molecular weight PAHs.

Biotransformation by the mixed function oxidase system in the fish liver can result

in the formation of carcinogenic and mutagenic intermediates, and exposure to

PAHs has been linked to the development of tumors in fish (Eisler, 1987). The

ability of fish to metabolize PAHs probably explains why benzo[a]pyrene

frequently is not detected or is found only at very low concentrations in fish from

areas heavily contaminated with PAHs (Varanasi and Gmur, 1980, 1981).

Sediment-associated PAHs can be accumulated by bottom-dwelling invertebrates

and fish (Eisler, 1987). For example, Great Lakes sediments containing elevated

levels of PAHs were reported by Eadie et al. (1983) to be the source of the body

burdens of the compounds in bottom-dwelling invertebrates. Similarly, Varanasi

et al. (1985) found that benzo[a]pyrene was accumulated in fish, amphipod

crustaceans, shrimp, and clams when estuarine sediment was the source of the

compound. Approximate tissue-to-sediment ratios were 0.6 to 1.2 for amphipods,

0.1 for clams, and 0.05 for fish and shrimp. Although fish and most crustaceans

evaluated to date have the mixed function oxidase system required for

biotransformation of PAHs, many molluscs lack this system and are unable to

metabolize PAHs efficiently (Varanasi et al., 1985). More important, PAHs induce

mixed function oxidase enzymes (and thus their own biotransformation) in fish

and other vertebrates, but not in molluscs and crustaceans (Stegeman and Lech,

1991). The resulting dramatic difference in biotransformation means that in PAHcontaminated waters, fish may show little or no accumulation of PAHs, while

bivalve molluscs and crustaceans are heavily contaminated. Varanasi et al.

(1985) ranked benzo[a]pyrene metabolism by aquatic organisms as follows: fish

> shrimp > amphipod crustaceans > clams. Half-lives for elimination of PAHs in

fish ranged from less than 2 days to 9 days (Niimi, 1987). NAS (1991) reported

that PAH contamination in bivalves has been found in all areas of the United

States. If PAHs are selected as a target analyte to be monitored at a site, primary

preference should be given to selection of a bivalve mollusc (clam, oyster,

mussel) as the target species, secondary preference should be given to a

crustacean (shrimp, lobster, crab) (if available), and finfish should be given the

lowest priority for selection as the target species. This ranking of the preferred

target species for PAH analysis assumes that a bivalve mollusc and crustacean

are available at the sampling site and that these species are eaten by the

consumer population of concern.

In 1993, three states (Massachusetts, Michigan, and Ohio) had issued advisories

for PAH contamination in finfish (RTI, 1993). As of 1998, there were five

advisories in effect in four states (Massachusetts, Michigan, Ohio, and
4. TARGET ANALYTES

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Washington) for PAHs (U.S. EPA, 1999c). Monitoring sites in industrial and

suburban/urban watersheds should be reviewed to identify current and historic

sites of waste incinerators, coal gasification facilities, petroleum refineries, and

creosote, coal tar, coal coking, and wood preservative facilities that are potential

sources for PAH releases to the environment. Sites of petroleum spills should also

be reviewed. 

The EPA and others have developed a relative potency estimate approach for the

PAHs (Nisbet and LaGoy, 1992; U.S. EPA, 1993c). Using this approach, the

cancer potency of 14 carcinogenic PAHs can be estimated based on their relative

potency to benzo[a]pyrene. Toxicity equivalence factors (TEF) for benzo[a]pyrene

and the other 14 PAHs based on carcinogenicity are discussed in Section 5.3.2.4.

Although several PAHs have been classified as probable human carcinogens

(Group B2), benzo[a]pyrene is the only PAH for which an oral CSF is currently

available in IRIS (1999). It is recommended that, in both screening and intensive

studies, tissue samples be analyzed for benzo[a]pyrene and the other 14 PAHs

for which TEFs are available and that the relative potencies given for these PAHs

(Nisbet and LaGoy, 1992; U.S. EPA, 1993c) be used to calculate a potency

equivalency concentration for each sample for comparison with the recommended

SVs for benzo[a]pyrene (see Section 5.3.2.4). 

4.3.6 Polychlorinated Biphenyls (Total)

PCBs are base/neutral compounds that are formed by the direct chlorination of

biphenyl. PCBs are closely related to many chlorinated hydrocarbon pesticides

(e.g., DDT, dieldrin, and aldrin) in their chemical, physical, and toxicologic

properties and in their widespread occurrence in the aquatic environment (Nimmo,

1985). There are 209 different PCB compounds, termed congeners, based on the

possible chlorine substitution patterns. In the United States, mixtures of various

PCB congeners were formulated for commercial use under the trade name

Aroclor on the basis of their percent chlorine content. For example, a common

PCB mixture, Aroclor 1254, has an average chlorine content of 54 percent by

weight (Nimmo, 1985).

Unlike the organochlorine pesticides, PCBs were never intended to be released

directly into the environment; most uses were in closed industrial systems.

Important properties of PCBs for industrial applications include thermal stability,

fire and oxidation resistance, and solubility in organic compounds (Hodges, 1977).

PCBs were used as insulating fluids in electrical transformers and capacitors, as

plasticizers, as lubricants, as fluids in vacuum pumps and compressors, and as

heat transfer and hydraulic fluids (Hodges, 1977; Nimmo, 1985). Although use

of PCBs as a dielectric fluid in transformers and capacitors was generally

considered a closed-system application, the uses of PCBs, especially during the

1960s, were broadly expanded to many open systems where losses to the

environment were likely. Heat transfer systems, hydraulic fluids in die cast

machines, and uses in specialty inks are examples of more open-ended
4. TARGET ANALYTES

4-48

applications that resulted in serious contamination in fish near industrial discharge

points (Hesse, 1976).

Although PCBs were once used extensively by industry, their production and use

in the United States were banned by the EPA in July 1979 (Miller, 1979). Prior

to 1979, the disposal of PCBs and PCB-containing equipment was not subject to

federal regulation. Prior to regulation, of the approximately 1.25 billion pounds

purchased by U.S. industry, 750 million pounds (60 percent) were still in use in

capacitors and transformers, 55 million pounds (4 percent) had been destroyed

by incineration or degraded in the environment, and over 450 million pounds (36

percent) were either in landfills or dumps or were available to biota via air, water,

soil, and sediments (Durfee et al., 1976).

PCBs are extremely persistent in the environment and are bioaccumulated

throughout the food chain (Eisler, 1986; Worthing, 1991). There is evidence that

PCB health risks increase with increased chlorination because more highly

chlorinated PCBs are retained more efficiently in fatty tissues (IRIS, 1999).

However, individual PCB congeners have widely varying potencies for producing

a variety of adverse biological effects including hepatotoxicity, cardiovascular

toxicity, developmental toxicity, immunotoxicity, neurotoxicity, and carcinogenicity.

The non-ortho-substituted coplanar PCB congeners, and some of the mono-orthosubstituted congeners, have been shown to exhibit "dioxin-like" effects (Golub et

al., 1991; Kimbrough and Jensen, 1989; McConnell, 1980; Poland and Knutson,

1982; Safe, 1985, 1990; Tilson et al., 1990; U.S. EPA 1993c; Van den Berg et al.,

1998). The neurotoxic effects of PCBs appear to be associated with some degree

of ortho-chlorine substitution. There is increasing evidence that many of the toxic

effects of PCBs result from alterations in hormonal function. Because PCBs can

act directly as hormonal agonists or antagonists, PCB mixtures may have

complex interactive effects in biological systems (Korach et al., 1988; Safe et al.,

1991; Shain et al., 1991; U.S. EPA, 1993c). Because of the lack of sufficient

toxicologic data, EPA has not developed quantitative estimates of health risk for

specific congeners; however, 12 dioxin-like congeners have been assigned TEFs

and may be evaluated as contributing to dioxin health risk (Van den Berg et al.,

1998). PCB mixtures have been classified as probable human carcinogens

(Group B2) (Appendix G) (IRIS, 1999; U.S. EPA, 1988a).

PCB mixtures have been shown to cause adverse developmental effects in

experimental animals (ATSDR, 1998b). Data are inconclusive in regard to

developmental effects in humans. Several studies in humans have suggested

that PCB exposure may cause adverse developmental effects in children and in

developing fetuses (ATSDR, 1998b) These include lower IQ scores (Jacobson

and Jacobson, 1996), low birth weight (Rylander et al., 1998), and lower behavior

assessment scores (Lonky et al., 1996). However, study limitations, including

lack of control for confounding variables, deficiencies in the general areas of

exposure assessment, selection of exposed and control subjects, and the

comparability of exposed and control samples obscured interpretation of these

results (ATSDR, 1998b).
4. TARGET ANALYTES

4-49

PCBs, total or as Aroclors, have been included in seven major fish contaminant

monitoring programs evaluated by the 1993 EPA Workgroup (Appendix E). A

summary of the U.S. Fish and Wildlife Service National Contaminants

Biomonitoring Program (NCBP) data from 1976 through 1984 indicated a

significant downward trend in the geometric mean concentration (wet weight

basis) of total PCBs (from 0.89 ppm in 1976 to 0.39 ppm in 1984); however, PCB

residues in fish tissue remain widespread, being detected at 91 percent of the

sites monitored in 1984 (Schmitt et al., 1990). Maximum total PCB tissue residue

concentrations during this same period also declined, from 70.6 ppm in 1976 to

6.7 ppm in 1984. Coinciding declines in tissue residue concentrations of three

Aroclors (1248, 1254, and 1260) were also observed. Kidwell et al. (1995)

conducted an analysis of all 1984-1985 data from the NCBP study on the three

Aroclors in bottom-feeding and predatory fish species. These authors reported

there was no significant difference in residues in these two trophic groups of fish

for Aroclor 1248 and 1254; however, there were significantly higher

concentrations of Aroclor 1260 in predator species as compared to bottom

feeders. Mean tissue concentrations of Aroclor 1248, 1254, and 1260 were 0.06

± 0.32, 0.21 ± 0.39, and 0.14 ± 0.24 ppm, respectively, for bottom feeders (e.g.,

carp, white suckers, and channel catfish) and 0.08 ± 0.31, 0.35 ± 0.69, and 0.23

± 0.38 ppm, respectively, for predator species (e.g., rainbow, brown, brook, and

lake trout, largemouth bass, and walleye).

Total PCBs also were detected at 91 percent of 374 sites surveyed in the EPA

National Study of Chemical Residues in Fish (NSCRF) (U.S. EPA, 1992c, 1992d).

Maximum, arithmetic mean, and median total PCB concentrations (wet weight)

reported were 124, 1.89, and 0.209 ppm, respectively. As is shown in Table 4-6,

the tri-, tetra-, penta-, hexa-, and heptachlorobiphenyls were detected in fish

tissue samples at >50 percent of the NSCRF sites. Mean tissue concentrations

were highest for the tetra- and pentachlorobiphenyls with concentrations of 0.696,

0.565, and 0.356 ppm, respectively. The median fish tissue concentrations were

highest for the hexa- followed by the pentachlorobiphenyls with concentrations of

0.077 and 0.072 ppm, respectively. 

With respect to sources of these compounds, PCBs were detected in all parts of

the country with the highest concentrations being associated with paper mills,

refinery/other industry sites, Superfund sites, wood preserving facilities, and

industrial/urban areas. Mean total PCB concentrations from the NSCRF study

were highest in bottom feeders (whole fish) such as carp (2.94 ppm), white sucker

(1.7 ppm), and channel catfish (1.3 ppm) as compared to predator species (fillet

samples) such as largemouth bass (0.23 ppm), smallmouth bass (0.5 ppm), and

walleye (0.37 ppm) (Kuehl et al., 1994). 

In 1993, PCB contamination in fish and shellfish resulted in the issuance of 328

advisories in 31 states and the U.S. territory of American Samoa (Figure 4-3)

(RTI, 1993). As of 1998, there were 679 advisories in effect in 36 states and the

U.S. territory of American Samoa for this compound (Figure 4-3) (U.S. EPA,

1999c.). In addition, two states (Indiana and New York) and the District of

Columbia had statewide advisories for PCBs in freshwater rivers and/or lakes. 
4. TARGET ANALYTES

4-50

One state, Connecticut, had an advisory for all its coastal estuarine waters (Long

Island Sound), and five states (Massachusetts, New Hampshire, New Jersey,

New York, and Rhode Island) had advisories in effect for all of their coastal

marine waters (U.S. EPA, 1999c). Monitoring sites in industrial and suburban/

urban watersheds should be reviewed to identify sites of historical Aroclor

production facilities, current and historic transformer manufacturing or refurbishing

facilities, current and historic landfill and Superfund sites, and current and historic

incineration or combustion facilities that are potential sources for PCB releases

to the environment. 

PCBs may be analyzed quantitatively as Aroclor equivalents, as homologue

groups, or as individual congeners. Historically, Aroclor analysis has been

performed by most laboratories. This procedure can, however, result in significant

error in determining total PCB concentrations (Schwartz et al., 1987; Cogliano,

1998; U.S. EPA, 1996) and in assessing the toxicologic significance of PCBs,

because it is based on the assumption that distribution of PCB congeners in

environmental samples and parent Aroclors is similar.

The distribution of PCB congeners in Aroclors is, in fact, altered considerably by

physical, chemical, and biological processes after release into the environment,

particularly when the process of biomagnification is involved (Norstrom, 1988;

Oliver and Niimi, 1988; Smith et al., 1990; U.S. EPA, 1996). Aquatic environmental studies indicate that the chlorine content of PCBs increases at higher

trophic levels (Bryan et al., 1987; Kubiak et al., 1989; Oliver and Niimi, 1988). 

Table 4-6. Summary of PCBs Detected in Fish Tissuea as Part of the

National Study of Chemical Residues in Fish

Congener group

% sites

where

detected Maximum Mean Median

Monochlorobiphenyl 13.8 0.235 0.001 ND

Dichlorobiphenyl 30.7 5.072 0.021 ND

Trichlorobiphenyl 57.5 18.344 0.150 0.002

Tetrachlorobiphenyl 72.4 60.764 0.696 0.023

Pentachlorobiphenyl 86.7 29.578 0.565 0.072

Hexachlorobiphenyl 88.7 8.862 0.356 0.077

Heptachlorobiphenyl 69.1 1.850 0.097 0.017

Octachlorobiphenyl 34.8 0.593 0.017 ND

Nonachlorobiphenyl 9.7 0.413 0.003 ND

Decachlorobiphenyl 3.3 0.038 0.001 0.003

Total PCBs* 91.4 ----- 1.898 0.209

* The sum of the concentrations of compounds with 1 to 10 chlorines.

a

 Concentrations are in ppm (μg/g) wet weight basis.

Source: U.S. EPA, 1992c, 1992d.
4. TARGET ANALYTES

4-51

Figure 4-3. States issuing fish and shellfish advisories for PCBs.
4. TARGET ANALYTES

4-52

The available data indicate that bioaccumulated PCBs are more toxic and more

persistent than the original Aroclor mixtures (Cogliano, 1998). Consequently,

analysis of homologue groups or congeners should provide a more accurate

determination of total PCB concentrations than Aroclor analysis. PCB concentrations derived from Aroclor methods may underestimate total PCBs. In one study,

the Delaware Department of National Resources and Environmental Control

(DDNREC) compared results of PCBs in six fish samples as determined by

Aroclor analysis (Method 608) and homologue analysis (Method 680) (Greene,

1992). On the average, the homologue method gave PCB estimates that were

230 percent higher than the results from the Aroclor method.

The major advantage to analyzing PCBs as Aroclor equivalents is that the

analysis is relatively inexpensive (approximately $200 - $500) compared to

analyzing PCBs as individual congeners (approximately $800-$2000). Another

disadvantage to analyzing PCBs as individual congeners is that the large number

of PCB congeners presents analytical difficulties. Quantitation of individual PCB

congeners is relatively time-consuming. EPA has not issued a standard method

for PCB congener analysis but has developed a draft method (1668) for dioxin-like

congeners (U.S. EPA 1997a). This method is likely to be revised to include the

capability to detect all 209 PCB congeners. Currently, only a few laboratories

have the capability or expertise to perform congener analyses. Both NOAA

(MacLeod et al., 1985; NOAA, 1989b) and the EPA Narragansett Research

Laboratory conduct PCB congener analyses. Some states currently conduct both

congener and Aroclor analysis; however, most states routinely perform only

Aroclor analysis. Analytical methods for congener analysis are discussed in the

following references: Cogliano, 1998; Huckins et al., 1988; Kannan et al., 1989;

Lake et al., 1995; MacLeod et al., 1985; Maack and Sonzogni, 1988; Mes and

Weber, 1989; NOAA, 1989b; Skerfving et al., 1994; Smith et al., 1990; Tanabe et

al., 1987; U.S. EPA, 1996. 

For the purposes of conducting a risk assessment to determine whether tissue

residues exceed potential levels of public health concern in fish and shellfish

monitoring programs, analysis of PCB congener or Aroclor equivalents is acceptable. However, because of their lower cost, Aroclor analyses may be the more

cost-effective method to use if a large number of samples are analyzed for PCB

contamination.

States are encouraged to develop the capability to perform PCB congener

analysis. When congener analysis is conducted, at a minimum the 18 congeners

recommended by NOAA (shown in Table 4-7) should be analyzed and summed

to determine a total PCB concentration according to the approach used by NOAA

(1989b). States may wish to consider including additional congeners based on

site-specific considerations. PCB congeners of potential environmental

importance identified by McFarland and Clarke (1989) and dioxin-like congeners

identified by Van den Berg et al. (1998) also are listed in Table 4-7. Lake et al.

(1995) and Oliver and Niimi (1988) included more than 80 congeners in their

analyses of PCB patterns in water, sediment, and aquatic organisms. A recent

study conducted by the DDNREC (Greene, 1999) analyzed for 75 congeners in
4. TARGET ANALYTES

4-53

Table 4-7. Polychlorinated Biphenyl (PCB) Congeners Recommended for

Quantitation as Potential Target Analytes

PCB Congenera,b NOAAc

McFarland and Clarke

(1989)

DioxinLike PCBsf Highest

priorityd Secondprioritye

2,4’ diCB 8

2,2’,5 triCB

2,4,4’ triCB

3,4,4’ triCB

18

28

18

37

2,2’3,5’ tetraCB

2,2’4,5’ tetraCB

2,2’,5,5’ tetraCB

2,3’,4,4’ tetraCB

2,3’,4’,5 tetraCB

2,4,4’,5 tetraCB

3,3’,4,4’ tetraCB

3,4,4’,5 tetraCB

44

52

66

77

77

44

49

52

70

74

81

77

81

2,2’,3,4,5’ pentaCB

2,2’,3,4’,5 pentaCB

2,2’,4,5,5’ pentaCB

2,3,3’,4,4’ pentaCB

2,3,4,4’,5 pentaCB

2,3’,4,4’,5 pentaCB

2,3’,4,4’,6 pentaCB

2’,3,4,4’,5 pentaCB

3,3’,4,4’,5 pentaCB

101

105

118

126

87

90

101

105

118

126

114

119

123

105

114

118

123

126

2,2’,3,3’,4,4’ hexaCB

2,2’,3,4,4’,5’ hexaCB

2,2’,3,5,5’,6 hexaCB

2,2’,4,4’,5,5’ hexaCB

2,3,3’,4,4’,5 hexaCB

2,3,3’,4,4’,5 hexaCB

2,3,3’,4,4’,6 hexaCB

2,3’,4,4’,5,5’ hexaCB

2,3’,4,4’,5’,6 hexaCB

3,3’,4,4’,5,5’ hexaCB

128

138

153

169

128

138

153

156

169

151

157

158

167

168

156

157

167

169

2,2’,3,3’,4,4’,5 heptaCB

2,2’,3,4,4’,5,5’ heptaCB

2,2’,3,4,4’,5’,6 heptaCB

2,2’,3,4,4’,6,6’ heptaCB

2,2’,3,4’,5,5’,6 heptaCB

2,3,3’,4,4’,5,5’ heptaCB

2,2’,3,3’,4,4’,5,6 octaCB

2,2’,3,3’,4,5’,6,6’ octaCB

170

180

187

170

180

183

184

195

187

189

201

189

2,2’,3,3’,4,4’,5,5’,6 nonaCB 206

2,2’,3,3’,4,4’,5,5’,6,6’ decaCB 209

a Congeners recommended for quantitation, from dichlorobiphenyl (diCB) through decachlorobiphenyl (decaCB). b Congeners are identified in each column by their International Union of Pure and Applied

Chemistry (IUPAC) number, as referenced in Ballschmitter and Zell (1980) and Mullin et al.

(1984). c EPA recommends that these 18 congeners be summed to determine total PCB concentration

(NOAA, 1989b). d PCB congeners having highest priority for potential environmental importance based on

potential for toxicity, frequency of occurrence in environmental samples, and relative

abundance in animal tissues. e Congeners having second priority for potential environmental importance based on potential

for toxicity, frequency of occurrence in environmental samples, and relative abundance in

animal tissues. f Van den Berg et al., 1998.
4. TARGET ANALYTES

4-54

fish tissue. Of the 75 congeners, 40 were detected in every fish sample and 20

other congeners were detected in at least half the samples. The DDNREC

concluded that a comprehensive target congener list is needed to account for total

PCBs in environmental samples because most of the congeners contributed less

than 5 percent of the total PCBs.

The EPA Office of Water recommends that PCBs be analyzed as either

congeners or Aroclors, with total PCB concentrations reported as the sum of the

individual congeners or the sum of the individual Aroclors. If a congener analysis

is conducted, the 12 dioxin-like congeners identified in Table 4-7 may be

evaluated separately as part of the dioxin risk (see Section 4.3.7). The

recommendation is intended to allow states flexibility in PCB analysis and to

encourage the continued development of reliable databases of PCB congener and

Aroclor equivalents concentrations in fish and shellfish tissue in order to increase

our understanding of the mechanisms of action and toxicities of these chemicals.

The rationale for, and the uncertainties of, this recommended approach are

discussed further in Section 5.3.2.6.

4.3.7 Dioxins and Dibenzofurans

Note: At this time, EPA’s Office of Research and Development is reevaluating the

potency of dioxins and dibenzofurans. Information provided here as well as

information in Section 5.3.2.7 related to calculating TEQs and SVs for dioxins/

furans has been modified since the second edition of this Volume 1 guidance was

published, but is subject to change pending the results of this reevaluation.

The polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are included as target analytes primarily because of the extreme

potency of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD). Extremely low

doses of this homologue have been found to elicit a wide range of toxic responses

in animals, including carcinogenicity, teratogenicity, fetotoxicity, reproductive

dysfunction, and immunotoxicity (U.S. EPA, 1987d). This compound is the most

potent animal carcinogen evaluated by EPA, and EPA has determined that there

is sufficient evidence to conclude that 2,3,7,8-TCDD is a probable human carcinogen (B2) (HEAST, 1997). Concern over the health effects of 2,3,7,8-TCDD

is increased because of its persistence in the environment and its high potential

to bioaccumulate (U.S. EPA, 1987d). As of 1998, the TEF value for

1,2,3,7,8-PeCDD was changed from 0.5 to 1.0, giving 1,2,3,7,8-PeCDD and

2,3,7,8-TCDD the same toxicity equivalency factor (Van den Berg et al., 1998).

1,2,3,7-8-PeCDD is also one of the congeners that is bioaccumulated by fish

(U.S. EPA, 1992c, 1992d).

Because dioxin/furan contamination is found in proximity to industrial sites (e.g.,

bleached kraft paper mills or facilities handling 2,4,5-trichlorophenoxyacetic acid

[2,4,5-T], 2,4,5-trichlorophenol [2,4,5-TCP], and/or silvex), and municipal or

industrial combustors and incinerators (U.S. EPA, 1987d), it is recommended that

each state agency responsible for monitoring include these compounds as target

analytes on a site-specific basis based on the presence of potential sources and
4. TARGET ANALYTES

4-55

results of any environmental (water, sediment, soil, air) monitoring performed in

areas adjacent to these sites. All states should maintain a current awareness of

potential dioxin/furan contamination, including contamination from the 12 coplanar

PCBs that exhibit dioxin-like effects.

Fifteen dioxin and dibenzofuran congeners have been included in two major fish

contaminant monitoring programs; however, one congener, 2,3,7,8-TCDD, has

been included in six national monitoring programs (Appendix E). Six dioxin

congeners and nine dibenzofuran congeners were measured in fish tissue

samples in the EPA National Study of Chemical Residues in Fish. The various

dioxin congeners were detected at 32 to 89 percent of the 388 sites surveyed,

while the furan congeners were detected at 1 to 89 percent of the 388 sites

surveyed (U.S. EPA, 1992c, 1992d). As shown in Table 4-8, the dioxin/furan

congeners detected at more than 50 percent of the sites included four CDD

compounds and three CDF compounds: 1,2,3,4,6,7,8 HpCDD (89 percent),

2,3,7,8 TCDF (89 percent), 2,3,7,8 TCDD (70 percent), 1,2,3,6,7,8 HxCDD

(69 percent), 2,3,4,7,8 PeCDF (64 percent), 1,2,3,4,6,7,8 HpCDF (54 percent),

and 1,2,3,7,8 PeCDD (54 percent). The most frequently detected CDD/CDF

compounds (1,2,3,4,6,7,8-HpCDD and 2,3,7,8-TCDF) were also detected at the

highest concentrations–249 ppt and 404 ppt (wet weight), respectively. The mean

concentrations of these two compounds were considerably lower, at 10.5 and

13.6 ppt, respectively. The dioxin congener (2,3,7,8-TCDD) believed to be one

of the two most toxic congeners to mammals was detected at 70 percent of the

sites at a maximum concentration of 204 ppt and a mean concentration of 6.8 ppt.

The other toxic congener, 1,2,3,7,8-PeCDD, was detected at 54 percent of the

sites at a maximum and mean concentration of 53.95 and 2.38 ppt, respectively.

The NSCRF data showed that pulp and paper mills using chlorine bleach pulp

were the dominant source of 2,3,7,8-TCDD and 2,3,7,8-TCDF and that these sites

had the highest median 2,3,7,8-TCDD concentrations (5.66 ppt), compared to

other source categories studied, including refinery/other industrial sites (1.82 ppt),

industrial/urban sites (1.40 ppt), Superfund sites (1.27 ppt), and background sites

(0.5 ppt). Source categories that had the highest 2,3,7,8-TCDD concentrations in

fish also had the highest TEQ values. It should be noted that OCDD and OCDF

were not analyzed in fish tissues because the TEFs were zero for these

compounds at the initiation of the NSCRF study. In 1989, TEFs for OCDD and

OCDFs were given a TEF value of 0.001. Therefore, TEQ values presented in

the NSCRF report may be underreported for samples collected at sites with

sources of OCDD/OCDF contamination (e.g., wood preservers) (U.S. EPA, 1992,

1992d). It is noted that the latest TEFs for OCDD and OCDF are 0.0001 (Van

den Berg et al., 1998) (see Table 5-6).

In 1993, 20 states had issued 67 fish advisories for dioxins/furans (Figure 4-4)

(RTI, 1993). As of 1998, there were 59 advisories in effect in 19 states for this

chemical contaminant (Figure 4-4) (U.S. EPA, 1999c). In addition, three states

(Maine, New Jersey, and New York) had dioxin advisories in effect for all coastal

marine waters (U.S. EPA, 1999c).
4. TARGET ANALYTES

4-56

Table 4-8. Summary of Dioxins/Furans Detected in Fish

Tissue as Part of the EPA National Study of Chemical Residues in Fisha

Congener

% Sites

where

detected Maximum Mean

Standard 

deviation Median

Dioxins

2,3,7,8-TCDD 70 203.6 6.89 19.41 1.38

1,2,3,7,8-PeCDD 54 53.95 2.38 4.34 0.93

1,2,3,4,7,8-HxCDD 32 37.56 1.67 2.39 1.24

1,2,3,6,7,8-HxCDD 69 100.9 4.30 9.25 1.32

1,2,3,7,8,9-HxCDD 38 24.76 1.16 1.74 0.69

1,2,3,4,6,7,8-HpCDD 89 249.1 10.52 25.30 2.83

Furans

2,3,7,8-TCDF 89 403.9 13.61 40.11 2.97

1,2,3,7,8-PeCDF 47 120.3 1.71 7.69 0.45

2,3,4,7,8-PeCDF 64 56.37 3.06 6.47 0.75

1,2,3,4,7,8-HxCDF 42 45.33 2.35 4.53 1.42

1,2,3,6,7,8-HxCDF 21 30.86 1.74 2.34 1.42

1,2,3,7,8,9-HxCDF 1 0.96b 1.22 0.41 1.38

2,3,4,6,7,8-HxCDF 32 19.3 1.24 1.51 0.98

1,2,3,4,6,7,8-HpCDF 54 58.3 1.91 4.41 0.72

1,2,3,4,7,8,9-HpCDF 4 2.57 1.24 0.33 1.30

EPA-TEQc NA 213 11.1 23.8 2.80

a Concentrations are given in picograms per gram (pg/g) or parts per trillion (ppt) by wet weight. 

The mean, median, and standard deviation were calculated using one-half the detection limit

for samples that were below the detection limit. In cases where multiple samples were

analyzed per site, the value used represents the highest concentration. b Detection limits were higher than the few quantified values for 1,2,3,4,7,8,9-HpCDF and

1,2,3,7,8,9-HxCDF. Maximum values listed are measured values. c This EPA study used TEF-89 toxicity weighting values but did not analyze concentrations of

octachlorodibenzo-p-dioxin or octachlorodibenzofurans in fish tissues; therefore, the TEQ

value does not include these two compounds or the 12 coplanar PCB congeners.

EPA = U.S. Environmental Protection Agency

HpCDD = Heptachlorodibenzo-p-dioxin

HpCDF = Heptachlorodibenzofuran

HxCDD = Hexachlorodibenzo-p-dioxin

HxCDF = Hexachlorodibenzofuran

NA = Not applicable

PeCDD = Pentachlorodibenzo-p-dioxin

PeCDF = Pentachlorodibenzofuran

TCDD = Tetrachlorodibenzo-p-dioxin

TCDF = Tetrachlorodibenzofuran

TEQ = Toxicity equivalency concentration.

Source: U.S. EPA, 1992c and 1992d.
4. TARGET ANALYTES

4-57

Figure 4-4. States issuing fish and shellfish advisories for dioxin/furans.
4. TARGET ANALYTES

4-58

Table 4-9. Dibenzo-p-Dioxins and Dibenzofurans Recommended

for Analysis as Target Analytes

Dioxins Furans

2,3,7,8-TCDD 2,3,7,8-TCDF 

1,2,3,7,8-PeCDD 1,2,3,7,8-PeCDF

2,3,4,7,8-PeCDF

1,2,3,4,7,8-HxCDD

1,2,3,6,7,8-HxCDD

1,2,3,7,8,9-HxCDD

1,2,3,4,7,8-HxCDF

1,2,3,6,7,8-HxCDF

1,2,3,7,8,9-HxCDF

2,3,4,6,7,8-HxCDF

1,2,3,4,6,7,8-HpCDD 1,2,3,4,6,7,8-HpCDF

1,2,3,4,7,8,9-HpCDF

OCDD OCDF

Source: Van den Berg et al., 1998.

Dioxins/furans should be considered for analysis primarily in suburban/urban and

industrial watersheds at sites of pulp and paper mills using a chlorine bleaching

process and at industrial sites where the following organic compounds have been

or are currently produced: herbicides (containing 2,4,5-trichlorophenoxy acids

and 2,4,5-trichlorophenol), silvex, hexachlorophene, pentachlorophenol, and

PCBs as well as at sites of municipal and industrial waste incinerators and

combustors (U.S. EPA, 1987d). EPA recommends that all of the 17 2,3,7,8-

substituted tetra- through octachlorinated dioxin and dibenzofuran congeners

shown in Table 4-9 as well as the 12 dioxin-like PCB congeners shown in

Table 4-7 be included as target analytes. 

4.4 TARGET ANALYTES UNDER EVALUATION

At present, the EPA Office of Water is evaluating one metal (lead) for possible

inclusion as a recommended target analyte in state fish and shellfish contaminant

monitoring programs. A toxicologic profile for this metal and the status of the

evaluation are provided in this section. Other contaminants will be evaluated and

may be recommended as target analytes as additional toxicologic data become

available.

Note: Any time a state independently deems that an analyte currently under

evaluation and/or other contaminants are of public health concern within its

jurisdiction, the state should include these contaminants in its fish and shellfish

contaminant monitoring program.

4.4.1 Lead

Lead is derived primarily from the mining and processing of limestone and

dolomite deposits, which are often sources of lead, zinc, and copper (May and

McKinney, 1981). It is also found as a minor component of coal. Historically, lead

has had a number of industrial uses, including use in paints, in solder used in

plumbing and food cans, and as a gasoline additive. In the past, the primary
4. TARGET ANALYTES

4-59

source of lead in the environment was the combustion of gasoline; however, use

of lead in U.S. gasoline has fallen sharply in recent years due to an EPA phasedown program to minimize the amount of lead in gasoline over time. By 1988, the

total lead usage in gasoline had been reduced to less than 1 percent of the

amount used in the peak year of 1970 (ATSDR, 1997). At present, lead is used

primarily in batteries, electric cable coverings, ammunition, electrical equipment,

and sound barriers. Currently, the major points of entry of lead into the

environment are from industrial processes, including metals processing, waste

disposal and recycling, and chemical manufacturing and from the leachates of

landfills (ATSDR, 1997; May and McKinney, 1981).

Lead has been included in five national monitoring programs (Appendix E). Lead

has been shown to bioaccumulate, with the organic forms, such as tetraethyl lead,

appearing to have the greatest potential for bioaccumulation in fish tissues. High

concentrations of lead have been found in marine bivalves and finfish from both

estuarine and marine waters (NOAA, 1987, 1989a). In 1984 and 1985, the U.S.

Fish and Wildlife Service collected 315 composite samples of whole fish from 109

stations nationwide as part of the National Contaminant Biomonitoring Program

(Schmitt and Brumbaugh, 1990). The authors reported that the maximum,

geometric mean, and 85th percentile concentrations for lead were 4.88, 0.11, and

0.22 ppm (wet weight), respectively. Lead concentrations in freshwater fish

declined significantly from a geometric mean concentration of 0.28 ppm in 1976

to 0.11 ppm in 1984. This trend has been attributed primarily to reductions in the

lead content of U.S. gasoline (Schmitt and Brumbaugh, 1990). Kidwell et al.

(1995) conducted an analysis of lead levels in tissues from bottom-feeding and

predatory fish using the 1984-1985 data from the NCBP study. These authors

reported that the mean lead tissue concentrations of 0.18 ± 0.37 ppm in bottom

feeders and 0.15 ± 0.43 ppm in predator fish were not significantly different. 

In 1993, three states (Massachusetts, Missouri, and Tennessee) and the U.S.

territory of American Samoa had fish advisories for lead contamination (RTI,

1993). As of 1998, there were 10 advisories in effect in four states (Hawaii,

Louisiana, Missouri, and Ohio) and the U.S. territory of American Samoa for this

heavy metal (U.S. EPA, 1999c).

Lead is particularly toxic to children and fetuses. Subtle neurobehavioral effects

(e.g., fine motor dysfunction, impaired concept formation, and altered behavior

profile) occur in children exposed to lead at concentrations that do not result in

clinical encephalopathy (ATSDR, 1997). A great deal of information on the health

effects of lead has been obtained through decades of medical observation and

scientific research. By comparison to most other environmental toxicants, the

degree of uncertainty about the health effects of lead is quite low. It appears that

some of these effects, particularly changes in the levels of certain blood enzymes

and in aspects of children's neurobehavioral development, may occur at blood

lead levels so low as to be essentially without a threshold. EPA's Reference Dose

(RfD) Work Group discussed inorganic lead (and lead compounds) in 1985 and

considered it inappropriate to develop an RfD for inorganic lead (IRIS, 1999).

Lead and its inorganic compounds have been classified as probable human
4. TARGET ANALYTES

4-60

carcinogens (B2) by EPA (IRIS, 1999). However, EPA has not derived a quantitative estimate of carcinogenic risk from oral exposure to lead because age,

health, nutritional status, body burden, and exposure duration influence the

absorption, release, and excretion of lead. In addition, current knowledge of lead

pharmacokinetics indicates that an estimate derived by standard procedures

would not truly describe the potential risk (IRIS, 1999).

Because of the lack of quantitative health risk assessment information for oral

exposure to inorganic lead, the EPA Office of Water has not included lead as a

recommended target analyte in fish and shellfish contaminant monitoring

programs at this time. Note: Because of the observation of virtually no-threshold

neurobehavioral developmental effects of lead in children, states should include

lead as a target analyte in fish and shellfish contaminant programs if there is any

evidence that this metal may be present at detectable levels in fish or shellfish

in their jurisdictional waters.
5. SCREENING VALUES FOR TARGET ANALYTES

5-1

SECTION 5 

SCREENING VALUES FOR TARGET ANALYTES

For the purpose of this guidance document, screening values are defined as

concentrations of target analytes in fish or shellfish tissue that are of potential

public health concern and that are used as threshold values against which levels

of contamination in similar tissue collected from the ambient environment can be

compared. Exceedance of these SVs should be taken as an indication that more

intensive site-specific monitoring and/or evaluation of human health risk should

be conducted.

The EPA-recommended risk-based method for developing SVs (U.S. EPA,

1989d) is described in this section. This method is considered to be appropriate

for protecting the health of fish and shellfish consumers for the following reasons

(Reinert et al., 1991):

• It gives full priority to protection of public health.

• It provides a direct link between fish consumption rate and risk levels (i.e.,

between dose and response).

• It generally leads to conservative estimates of increased risk.

• It is designed for protection of consumers of locally caught fish and shellfish,

including susceptible populations such as sport and subsistence fishers who

are at potentially greater risk than the general adult population because they

tend to consume greater quantities of fish and because they frequently fish

the same sites repeatedly.

At this time, the EPA Office of Water is recommending use of this method

because it is the basis for developing current water quality criteria. A detailed

discussion of the flexibility of the EPA risk-based method and the use of EPA’s

SVs as compared to FDA action levels is provided in Section 1.2. Further discussion of the EPA Office of Water risk-based approach, including a detailed

description of the four steps involved in risk assessment (hazard identification,

dose-response assessment, exposure assessment, and risk characterization) is

provided in the second guidance document in this series, Volume 2: Risk

Assessment and Fish Consumption Limits. 

5.1 GENERAL EQUATIONS FOR CALCULATING SCREENING VALUES

Risk-based SVs are derived from the general model for calculating the effective

ingested dose of a chemical m (Em) (U.S. EPA, 1989d):
5. SCREENING VALUES FOR TARGET ANALYTES

5-2

Em = (Cm • CR • Xm) / BW (5-1)

where

Em = Effective ingested dose of chemical m in the population of concern

averaged over a 70-yr lifetime (mg/kg-d)

Cm = Concentration of chemical m in the edible portion of the species of

interest (mg/kg; ppm)

CR = Mean daily consumption rate of the species of interest by the general

population or subpopulation of concern averaged over a 70-yr lifetime

(kg/d)

Xm = Relative absorption coefficient, or the ratio of human absorption

efficiency to test animal absorption efficiency for chemical m

(dimensionless)

BW = Mean body weight of the general population or subpopulation of

concern (kg).

Using this model, the SV for the chemical m (SVm) is equal to Cm when the

appropriate measure of toxicologic potency of the chemical m (Pm) is substituted

for Em. Rearrangement of Equation 5-1, with these substitutions, gives

SVm = (Pm • BW) / (CR • Xm) (5-2)

where

Pm = Toxicologic potency for chemical m; the effective ingested dose of

chemical m associated with a specified level of health risk as

estimated from dose-response studies; dose-response variable.

In most instances, relative absorption coefficients (Xm) are assumed to be 1.0

(i.e., human absorption efficiency is assumed to be equal to that of the test

animal), so that 

SVm = (Pm • BW) / CR . (5-3)

However, if Xm is known, Equation 5-2 should be used to calculate SVm.

Dose-response variables for noncarcinogens and carcinogens are defined in

Sections 5.1.1 and 5.1.2, respectively. These variables are based on an assessment of the occurrence of a critical toxic or carcinogenic effect via a specific route

of exposure (i.e., ingestion, inhalation, dermal contact). Oral dose-response

variables for the recommended target analytes are given in Appendix G.

Because of the fundamental differences between the noncarcinogenic and

carcinogenic dose-response variables used in the EPA risk-based method, SVs
5. SCREENING VALUES FOR TARGET ANALYTES

5-3

must be calculated separately for noncarcinogens and potential carcinogens as

shown in the following subsections.

5.1.1 Noncarcinogens

The dose-response variable for noncarcinogens is the reference dose. The RfD

is an estimate of a daily exposure to the human population (including sensitive

subpopulations) that is likely to be without appreciable risk of deleterious effects

during a lifetime. The RfD is derived by applying uncertainty or modifying factors

to a subthreshold dose (i.e., lowest observed adverse effects level [LOAEL] if the

no observed adverse effect level [NOAEL] is indeterminate) observed in chronic

animal bioassays. These uncertainty or modifying factors range from 1 to 10 for

each factor and are used to account for uncertainties in:

• Sensitivity differences among human subpopulations

• Interspecies extrapolation from animal data to humans

• Short-term to lifetime exposure extrapolation from less-than-chronic results

on animals to humans when no long-term human data are available

• Deriving an RfD from a LOAEL instead of a NOAEL

• Incomplete or inadequate toxicity or pharmacokinetic databases.

The uncertainty (UF) and modifying (MF) factors are multiplied to obtain a final

UF•MF value. This factor is divided into the NOAEL or LOAEL to derive the RfD

(Barnes and Dawson, 1988; U.S. EPA, 1989d).

The following equation should be used to calculate SVs for noncarcinogens:

SVn = (RfD • BW)/CR (5-4)

where

SVn = Screening value for a noncarcinogen (mg/kg; ppm)

RfD = Oral reference dose (mg/kg-d)

and BW and CR are defined as in Equation 5-1.

5.1.2 Carcinogens

According to The Risk Assessment Guidelines of 1986 (U.S. EPA, 1987f), the

default model for low-dose extrapolation of carcinogens is a version (GLOBAL

86) of the linearized multistage no-threshold model developed by Crump et al.

(1976). This extrapolation procedure provides an upper 95 percent bound risk

estimate (referred to as a q1*), which is considered by some to be a conservative

estimate of cancer risk. Other extrapolation procedures may be used when

justified by the data.

Screening values for carcinogens are derived from: (1) a carcinogenicity potency

factor or cancer slope factor, which is generally an upper bound risk estimate;

and (2) a risk level (RL), an assigned level of maximum acceptable individual
5. SCREENING VALUES FOR TARGET ANALYTES

5-4

lifetime risk (e.g., RL = 10-5 for a level of risk not to exceed one excess case of

cancer per 100,000 individuals exposed over a 70-yr lifetime) (U.S. EPA, 1997b).

The following equation should be used to calculate SVs for carcinogens:

SVc = [(RL / CSF) • BW] / CR (5-5)

where

SVc = Screening value for a carcinogen (mg/kg; ppm)

RL = Maximum acceptable risk level (dimensionless)

CSF = Oral cancer slope factor (mg/kg-d)-1

and BW and CR are defined as in Equation 5-1.

5.1.3 Recommended Values for Variables in Screening Value Equations

The default values for variables used in Equations 5-4 and 5-5 to calculate SVs

are based on assumptions for the general adult population. These default values

are consistent with values included in the Methodology for Deriving Ambient

Water Quality Criteria for the Protection of Human Health (2000) (EPA-822-B-00-

004). For risk management purposes (e.g., to protect sensitive populations such

as pregnant and nursing women), states may choose to use alternative values

for consumption rates, etc. different from those recommended in this section. 

5.1.3.1 Dose-Response Variables—

EPA has developed oral RfDs and/or CSFs for all of the recommended target

analytes in Section 4 (see Appendix G). These are maintained in the EPA

Integrated Risk Information System (IRIS, 1999), an electronic database

containing health risk and EPA regulatory information on approximately 400

different chemicals. IRIS is available online at:

http://www.epa.gov/iris/subst/index.html

The IRIS RfDs and CSFs are reviewed regularly and updated as necessary when

new or more reliable information on the toxic or carcinogenic potency of

chemicals becomes available.

When IRIS values for oral RFDs and CSFs are available, they should be used to

calculate SVs for target analytes from Equations 5-4 and 5-5, respectively. It is

important that the most current IRIS values for oral RfDs and CSFs be used to

calculate SVs for target analytes unless otherwise recommended.

In cases where IRIS values for oral RFDs or CSFs are not available for

calculating SVs for target analytes, estimates of these variables may be derived

from the most recent water quality criteria (U.S. EPA, 1992e) according to

procedures described in U.S. EPA (1991a, p. IV-12), or from the Classification
5. SCREENING VALUES FOR TARGET ANALYTES

5-5

Table 5-1. Recommended Values for Mean Body Weights (BWs)

and Fish Consumption Rates (CRs) for Selected Subpopulations

Variable Recommended value Subpopulation

BW 70 kg

78 kg

65 kg

12 kg

17 kg

25 kg

36 kg

51 kg

61 kg

All adults (U.S. EPA, 1999a)

Adult males (U.S. EPA, 1985b, 1990a)

Adult females (U.S. EPA, 1985b, 1990a)

Children <3 yr (U.S. EPA, 1985b, 1990a)

Children 3 to <6 yr (U.S. EPA, 1985b, 1990a)

Children 6 to <9 yr (U.S. EPA, 1985b, 1990a)

Children 9 to <12 yr (U.S. EPA, 1985b, 1990a)

Children 12 to <15 yr (U.S. EPA, 1985b, 1990a)

Children 15 to <18 yr (U.S. EPA, 1985b, 1990a)

CRa 17.5 g/d (0.0175 kg/d) Estimate of the 90th percentile of recreational or

sport fishers (USDA/ARS, 1998) and of the

average consumption of uncooked fish and

shellfish from estuarine and fresh waters by

recreational fishers (U.S. EPA, 2000c)

142.4 g/d (0.1424 kg/d) Estimate of the 99th percentile of subsistence

fishers (USDA/ARS, 1998) and of the average

consumption of uncooked fish and shellfish from

estuarine and fresh waters by subsistence fishers

(U.S. EPA, 2000c)

a These are recommended default consumption rates only. Note: When local consumption

rate data are available for recreational and subsistence fishers, they should be used to

calculate SVs for noncarcinogens and carcinogens by subsistence fishers, as described in

Sections 5.1.1 and 5.1.2, respectively.

List of Chemicals Evaluated for Carcinogenicity Potential (U.S. EPA 1999b) from

the Office of Pesticide Programs Health Effects Division.

5.1.3.2 Body Weight and Consumption Rate—

Values for the variables BW and CR in Equations 5-4 and 5-5 are given in

Table 5-1 for various subpopulations including recreational and subsistence

fishers. Note: In this third edition of this document, EPA’s Office of Water uses

a BW of 70 kg, a default CR of 17.5 g/d to calculate the SV for the general

populations and recreational fishers, and a default CR of 142.4 g/d to calculate

the SV for subsistence fishers. The CR values have been revised since the

release of the previous edition.

The default CR of 6.5 g/d used in the previous edition of Volume I was based on

data from a fish consumption survey conducted in 1973 and 1974 by the National

Purchase Diaries and funded by the Tuna Institute. This value represented the

estimated mean per capita freshwater/estuarine finfish and shellfish consumption

rate for the general U.S. population (Jacobs et al., 1998). This value has been

revised based on new data from the combined 1994, 1995, and 1996 Continuing

Survey of Food Intake by Individuals (CSFII) survey (USDA/ARS, 1998). The
5. SCREENING VALUES FOR TARGET ANALYTES

5-6

CSFII survey is a national food consumption survey conducted by the U.S.

Department of Agriculture, consisting of multistage, stratified-cluster area

probability samples from all states except Alaska and Hawaii. 

These data are collected over 3 consecutive days. On the first day of the survey,

participants give information to an in-home interviewer, and on the second and

third days, data are taken from self-administered dietary records. Meals

consumed both at home and away from home are recorded. Average daily

individual consumptions of fish in a given fish-by-habitat category were calculated

by summing the amount of fish eaten by the individual across 3 reporting days

for all fish-related food codes in a given fish-by-habitat category. The total

individual consumption was then divided by three to obtain an average daily

consumption rate. The 3-day individual food consumption data collection period

is one during which a majority of sampled individuals did not consume any finfish

or shellfish. The nonconsumption of finfish or shellfish by a majority of

individuals, combined with consumption data from high-end consumers, resulted

in a wide range of observed fish consumption rates. This range of fish

consumption data would tend to produce distributions of fish consumption with

larger variances than would be associated with a longer survey period, such as

30 days. The larger variances would reflect greater dispersion, which results in

larger upper-percentile estimates, as well as upper confidence intervals

associated with parameter estimates. It follows that estimates of the upper

percentiles (90th and 99th percentiles) of per capita fish consumption based on 3

days of data will be consecutive with regard to risk (U.S. EPA, 1998a).

If states and tribes do not have site-specific fish consumption information

concerning their recreational and subsistence fishers, it is EPA’s preference that

they use as fish intake assumptions the default values from the most recent

1994-1996 CSFII study (USDA/ARS, 1998). The fish consumption default

values of 17.5 g/d for the general adult population and recreational fishers and

142.4 g/d for subsistence fishers used in this document are representative of fish

intake for these different population groups. These values are based on risk

management decisions that EPA has made after evaluating numerous fish

consumption surveys (U.S. EPA, 2000c). These default values represent the

uncooked weight intake of freshwater/estuarine finfish and shellfish. EPA

recognizes the data gaps and uncertainties associated with the analysis of the

1994-1996 CSFII survey conducted in the process of making its default

consumption rate recommendations. The estimated mean of freshwater/estuarine

fish ingestion for adults is 7.50 g/d, and the median is 0 g/d. The estimated 90th

percentile is 17.53 g/d; the estimated 95th percentile is 49.59 g/d; and the

estimated 99th percentile is 142.41 g/d. The median value of 0 g/d may reflect

the portion of individuals in the population who never eat fish as well as the

limited reporting period (2 days) over which intake was actually measured. By

applying as a default consumption rate the 17.5-g/d value for the general adult

population, EPA intends to select a consumption rate that is protective of the

majority of the population (the 90th percentile of consumers and nonconsumers

according to the 1994-1996 CSFII survey data). EPA further considers this rate

to be indicative of the average consumption among recreational fishers based on
5. SCREENING VALUES FOR TARGET ANALYTES

5-7

averages in the studies reviewed (U.S. EPA, 2000c). Similarly, EPA believes that

the assumption of 142.4 g/d is within the range of average consumption

estimates for subsistence fishers based on the studies reviewed. Experts at a

1992 National Water Quality Workshop acknowledged, however, that the

national survey high-end values are representative of average rates for highly

exposed groups such as subsistence fishers, specific ethnic groups, or other

high-risk populations. EPA is aware that some local and regional studies indicate

greater fish consumption among Native Americans, Pacific Asian Americans, and

other subsistence consumers and recommends the use of those studies in

appropriate cases. States and tribes have the flexibility to choose fish

consumption rates higher than an average value for these populations groups.

If a state has not identified a separate well-defined population of high-end

consumers and believes that the national data from the 1994-1996 CSFII are

representative, they may choose these consumption rates.

With respect to consumption rates, EPA recommends that states always evaluate

any type of consumption pattern they believe could reasonably be occurring at

a site. Evaluating additional consumption rates involves calculating additional

SVs only and does not add to sampling or analytical costs.

EPA has published a review and analysis of survey methods that can be used

by states to determine fish and shellfish consumption rates of local populations

(U.S. EPA, 1992b, 1998b). States should consult these documents to ensure

that appropriate values are selected to calculate SVs for site-specific exposure

scenarios. 

For any given population, there can be a sensitive subpopulation composed of

individuals who may be at higher-than-average risk due to their increased

exposure or their increased sensitivity to a contaminant or both. For Native

American subsistence fishers, there are several exposure issues of concern that

should be addressed as part of a comprehensive exposure assessment: 

• Consumption rates and dietary preferences. Harris and Harper (1997)

surveyed traditional tribal members in Oregon with a subsistence lifestyle and

determined a consumption rate of 540 g/d, which included fresh, dried, and

smoked fish. They also confirmed that the parts of the fish (heads, fins, tails,

skeleton, and eggs) eaten by this group were not typically eaten by other

groups. Another study conducted of four tribes in the Northwest that also

surveyed tribal members in Oregon but did not target subsistence fishers,

reported a 99th percentile ingestion rate of 390 g/d for tribal members

(CRITFC, 1994). These consumption rates are much higher than the default

consumption rates provided in this document for subsistence fishers and

emphasize the need for identifying the consumption rate of the Native

American subsistence population of concern. 

• Community characteristics - It is important to consider family-specific

fishing patterns in any exposure scenario, and attention should be paid to the

role of the fishing family with respect to the tribal distribution of fish, the
5. SCREENING VALUES FOR TARGET ANALYTES

5-8

sharing ethic, and providing fish for ceremonial religious events. Entire

communities are exposed if fish are contaminated, and the community

contaminant burden as a whole must be considered, not just the maximally

exposed individual.

• Multiple contaminant exposure - Multiple contaminant exposure is significant for Native American subsistence fishers. A large number of

contaminants are often detected in fish tissues and their combined risk

associated with the higher consumption rates and dietary preferences for

certain fish parts could be very high even if individual contaminants do not

exceed the EPA reference dose (Harper and Harris, 1999).

• Other exposure pathways - For Native American subsistence fishers,

overall exposure to a contaminant may be underestimated if it fails to take

into account nonfood uses of fish and other animal parts that may contribute

to overall exposure, such as using teeth and bones for decorations and

whistles, animal skins for clothing, and rendered fish belly fat for body paint

(Harper and Harris, 1999). If other wildlife species (e.g., feral mammals,

turtles, waterfowl) that also live in or drink from the contaminated waterbody

are eaten, or if the contaminated water is used for irrigation of crops or for

livestock watering or human drinking water, the relative source contribution

of these other pathways of exposure must also be considered. As with fish

and wild game, plants are used by Native Americans for more than just

nutrition. Daily cleaning, preparation, and consumption of plants and crafting

of plant materials into household goods occurs throughout the year (Harris

and Harper, 1997).

As in the general population, increased sensitivity to a chemical contaminant for

Native Americans can result from factors such as an individual’s underlying

health status and medications, baseline dietary composition and quality,

genetics, socioeconomic status, access to health care, quality of replacement

protein, age, gender, pregnancy, and lactation. These factors are only partially

considered in the uncertainty factor(s) used to develop the RfD (Harper and

Harris, 1999). 

Other important issues that need to be considered concern risk characterization

and risk management. For Native American subsistence fishers, the use of an

acceptable risk level of 1 in 100,000 (10-5) may not be acceptable to all tribes.

Each tribe has the right to decide for themselves what an acceptable level of risk

is, and, in some cases, it may be zero risk (zero discharge) to protect cultural

resources and uses. Ecological well-being or health is another key issue. Human

and ecological health are connected in many ways and the ripple effects are

often not recognized. For example, human health may be affected by injury to the

environment, which affects the economy and the culture (Harper and Harris,

1999). 

Native American subsistence fishers should be treated as a special high-risk

group of fish consumers distinct from fishers in the general population and
5. SCREENING VALUES FOR TARGET ANALYTES

5-9

distinct even from other Native American fish consumers living in more

suburbanized communities. Table 5-2 compares fish consumption rates for

various fisher populations within the general population and in several surveys

of specific Native American tribal populations. EPA currently recommends

default fish consumption rates of 17.5 g/d for the general and recreational fishers

and 142.4 g/d for subsistence fishers. However, the tribal population fish

consumption studies show that some Native American tribal members living in

river-based communities (CRITFC, 1994) eat from 3 to 22 times more fish (from

59 g/d up to 390 g/d) than do recreational fishers, but that traditional Native

American subsistence fishing families may eat up to 30 times more fish, almost

1.2 lb/d (540 g/d) (Harris and Harper, 1997). The fish consumption rate from

Harris and Harper (1997) for Native American subsistence fishers is also 3.8

times higher than the EPA default consumption rate for subsistence fishers

(142.4 g/d) in the general population. The difference in fish consumption is due

to the fact that the Native American subsistence fisher’s lifestyle is not the same

as a recreational fisher’s lifestyle with additional fish consumption added, nor is

it the same as the “average” Native American tribal member living in a fairly

suburbanized tribal community. In addition to exposures from direct consumption

of contaminated fish, Native American subsistence fishers also receive more

exposure to the water and sediments associated with catching and preparing fish

and possibly from drinking more unfiltered river water than more suburbanized

tribal community members as well. The Native American subsistence fishing

population should be treated as a separate group with a unique lifestyle, distinct

from recreational and subsistence fishers in the general U.S. population and also

distinct from other Native American fisher populations. 

5.1.3.3 Risk Level (RL)—

In this guidance document, EPA’s Office of Water uses an RL of 10-5 to calculate

screening values for the general adult population. However, states have the

flexibility to choose to use an appropriate RL value typically ranging from 10-4 to

10-7. This is the range of risk levels employed in various U.S. EPA programs.

Selection of the appropriate RL is a risk management decision that is made by

the state.

5.2 SCREENING VALUES FOR TARGET ANALYTES

Target analyte SVs, and the dose-response variables used to calculate them, are

given in Tables 5-3 and 5-4. The SVs are provided as default values for the

states to use when site-specific information on variables such as consumption

rates are not available for local recreational or subsistence fisher populations. 
5. SCREENING VALUES FOR TARGET ANALYTES

5-10

Table 5-2. Fish Consumption Rates for Various Fisher Populations

Source

Recreational

Fishers (g/d)

Subsistence

Fishers (g/d) 

Native American

Subsistence

Fishers (g/d) Native Americans (g/d)

Basis for Consumption

Rate

U.S. EPA 17.5 a142.4 a70 (mean) b

170 (95th

percentile)b

NA Fish consumption rate from

1994 and 1996 Continuing

Survey of Food Intake by

Individuals (CSFII)

Harris and

Harper

(1997)

NA NA 540 (fresh,

smoked and

dried)

NA Surveyed members of the

Confederated Tribes of the

Umatilla Indian Reservation 

CRITFC

(1994)

NA NA NA 59 (mean)

170 (95th percentile)

390 (99th percentile)

Surveyed members of the

Umatilla, Nez Perce,

Yakama, and Warm Springs

Tribes

Toy et al.

(1996)

NA NA NA 53 (median, males)

34 (median, females )

66 (median, males)

25 (median, females) 

Surveyed members of the

Tulalip Tribe

Surveyed members of the 

Squaxin Island Tribe

a These values were revised in this 3rd edition of Volume 1 of this series (USDA/ARS, 1998) 

b These values are from EPA’s Exposure Factors Handbook (U.S. EPA, 1997b) 

These SVs were calculated from Equations 5-4 or 5-5 using the following values

for BW, CR, and RL and the most current IRIS values for oral RfDs and CSFs

(IRIS, 1999) unless otherwise noted:

C For noncarcinogens:

BW = 70 kg, average adult body weight

CR = 17.5 g/d (0.0175 kg/d), estimate of average consumption of

uncooked fish and shellfish from estuarine and fresh waters by

recreational fishers, or 

= 142.4 g/d (0.1424 kg/d), estimate of average consumption of

uncooked fish and shellfish from estuarine and freshwaters by

subsistence fishers. 

C For carcinogens:

BW and CR, as above

RL = 10-5, a risk level corresponding to one excess case of cancer per

100,000 individuals exposed over a 70-yr lifetime.

If both oral RfD and CSF values are available for a given target analyte, SVs for

both noncarcinogenic and carcinogenic effects are listed in Table 5-2 for recreational fishers and Table 5-3 for subsistence fishers. Unless otherwise indicated,
5. SCREENING VALUES FOR TARGET ANALYTES

5-11

Table 5-3. Dose-Response Variables and Recommended Screening Values (SVs) for 

Target Analytes - Recreational Fishersa

Target analyte

Noncarcinogens

RfD (mg/kg-d)

Carcinogens

CSF (mg/kg-d)-1

SVb (ppm)

Noncarcinogensb

Carcinogensb

(RL=10-5)

Metals

Arsenic (inorganic)c3 x 10-4 1.5 1.2 0.026

Cadmium 1 x 10-3 NA 4.0 -

Mercury (methylmercury)d1 x 10-4 NA 0.4 -

Selenium 5 x 10-3 NA 20 -

Tributyltine3 x 10-4 NA 1.2 -

Organochlorine Pesticides

Total chlordane (sum of cis- and transchlordane, cis- and trans-nonachlor, and

oxychlordane)f

5 x 10-4 0.35 2.0 0.114

Total DDT (sum of 4,4'- and 2,4'- isomers of

DDT, DDE, and DDD)g

5 x 10-4 0.34 2.0 0.117

Dicofolh4 x 10-4 NAi1.6 2.5

Dieldrin 5 x 10-5 16 0.2 2.50 x 10-3

Endosulfan (I and II)j6 x 10-3 NA 24 -

Endrin 3 x 10-4 NA 1.2 -

Heptachlor epoxide 1.3 x 10-5 9.1 5.2 x 10-2 4.39 x 10-3

Hexachlorobenzene 8 x 10-4 1.6 3.2 2.50 x 10-2

Lindane (g-hexachlorocyclohexane;

g-HCH)k

3 x 10-4 1.3 1.2 3.07 x 10-2

Mirex 2 x 10-4 NAl0.8 -

Toxaphenej,m 2.5 x 10-4 1.1 1.0 3.63 x 10-2

Organophosphate Pesticides

Chlorpyrifosn3 x 10-4 NA 1.2 -

Diazinono7 x 10-4 NA 2.8 -

Disulfoton 4 x 10-5 NA 0.16 -

Ethion 5 x 10-4 NA 2.0 -

Terbufosp2 x 10-5 NA 0.08 -

Chlorophenoxy Herbicides

Oxyfluorfenq3 x 10-3 7.32 x 10-2 12 5.46 x 10-1

PAHsr NA 7.3 - 5.47 x 10-3

PCBs

Total PCBss2 x 10-5 2.0 0.08 0.02

Dioxins/furanst NA 1.56 x 105- 2.56 x 10-7

NA = Not available in EPA’s Integrated Risk

Information System (IRIS, 1999).

DDD = p,p’-dichlorodiphenyldichloroethane

DDT = p,p’-dichlorodiphenyltrichloroethane

DDE = p,p’-dichlorodiphenlydichloroethylene

PAH = Polycyclic aromatic hydrocarbon

PCB = Polychlorinated biphenyl

RfD = Oral reference dose (mg/kg-d)

CSF = Cancer slope factor (mg/kg-d)-1
5. SCREENING VALUES FOR TARGET ANALYTES

5-12

Table 5-3. (continued)

a Based on fish consumption rate of 17.5 g/d, 70kg body weight and, for carcinogens, 10-5 risk level and 70-yr lifetime. Unless otherwise

noted, values listed are the most current oral RfDs and CSF in EPA’s IRIS database (IRIS, 1999). 

b The shaded screening value (SV) is the recommended SV for each target analyte. States should note that the screening values listed may

be below analytical detection limits achievable for some of the target analytes. Please see Table 8-4 for detection limits.

c Total inorganic arsenic rather than total arsenic should be determined.

d Because most mercury in fish and shellfish tissue is present primarily as methylmercury (NAS, 1991;Tollefson, 1989) and because of the

relatively high cost of analyzing for methylmercury, it is recommended that total mercury be analyzed and the conservative assumption be

made that all mercury is present as methylmercury. This approach is deemed to be most protective of human health and most cost-effective.

The National Academy of Sciences conducted an independent assessment of the RfD for methylmercury. They concluded that “On the

basis of its evaluation, the committee’s consensus is that the value of EPA’s current RfD for methylmercury, 0.1Fg/kg per day, is a

scientifically justifiable level for the protection of human health”.

e The RfD value listed is for tributyltin oxide (IRIS, 1999).

f The RfD and CSF values listed are derived from studies using technical-grade chlordane (IRIS, 1999) for the cis- and trans-chlordane

isomers or the major chlordane metabolite, oxychlordane, or for the chlordane impurities cis- and trans-nonachlor. It is recommended that

total chlordane be determined by summing the concentrations of cis- and trans-chlordane, cis- and trans-nonachlor, and oxychlordane.

g The RfD value listed is for DDT. The CSF value (0.34) is for total DDT sum of DDT, DDE and DDD); the CSF value for DDD is 0.24. 

It is recommended that the total concentration of DDT include the 2,4'- and 4,4'-isomers of DDT and its metabolites, DDE and DDD. 

h The RfD value is from Office of Pesticide Programs Reregistration Eligibility Decision (RED) for Dicofol (EPA, 1998c).

i The CSF for dicofol was withdrawn from IRIS pending further review by the CRAVE Agency Work Group (IRIS, 1999).

j The RfD value listed is from the Office of Pesticide Program’s Reference Dose Tracking Report (U.S. EPA, 1997).

k

IRIS (1999) has not provided a CSF for lindane. The CSF value listed for lindane was calculated from the water quality criteria (0.063 mg/L)

(U.S. EPA, 1992f).

l No CSF or cancer classification is available for mirex. This compound is undergoing further review by the CRAVE Agency Work Group

(IRIS, 1999)

m The RfD value has been agreed upon by the Office of Pesticide Programs and the Office of Water.

n Because of the potential for adverse neurological developmental effects from chlorpyrifos, EPA recommends the use of a Population

Adjusted Dose (PAD) of 3 x 10-5 for infants, children under the age of 6 years, and women ages 13 to 50 years (U.S. EPA, 2000b).

o The RfD value is from a memorandum dated April 1, 1998, Diazinon:-Report of the Hazard Identification Assessment Review Committee.

HED Doc. No. 012558.

p The RfD value listed is from a memorandum dated September 25, 1997; Terbufos-FQPA Requirement- Report of the Hazard Idenification

Review.

q The CSF value is from the Office of Pesticide Programs List of Chemicals Evaluated for Carcinogenic Potential (U.S. EPA, 1999b).

r The CSF value listed is for benzo[a]pyrene. Values for other PAHs are not currently available in IRIS (1999). It is recommended that tissue

samples be analyzed for benzo[a]pyrene and 14 other PAHs, and that the order-of-magnitude relative potencies given for these PAHs

(Nisbet and LaGoy, 1992; U.S. EPA, 1993c) be used to calculate a potency equivalency concentration (PEC) for each sample (see Section

5.3.2.4). 

s Total PCBs may be determined as the sum of congeners or Aroclors. The RfD is based on Aroclor 1254 and should be applied to total

PCBs. The CSF is based on a carcinogenicity assessment of Aroclors 1260, 1254, 1242, and 1016. The CSF presented is the upperbound slope factor for food chain exposure. The central estimate is 1.0 (IRIS, 1999). 

t The CSF value listed is for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (HEAST, 1997). It is recommended that the 17 2,3,7,8-substituted

tetra- through octa-chlorinated dibenzo-p-dioxins and dibenzofurans and the 12 dioxin-like PCBs be determined and a toxicity-weighted

total concentration be calculated for each sample, using the method for estimating toxicity equivalency concentrations (TEQs) (Van den

Berg et al., 1998). 
5. SCREENING VALUES FOR TARGET ANALYTES

5-13

Table 5-4. Dose-Response Variables and Recommended Screening Values (SVs) for

Target Analytes - Subsistence Fishersa

Target analyte

Noncarcinogens

RfD (mg/kg-d)

Carcinogens

CSF (mg/kg-d)-1

SVb (ppm)

Noncarcinogensb

Carcinogensb

(RL=10-5)

Metals

Arsenic (inorganic)c3 x 10-4 1.5 0.147 3.27 x 10-3

Cadmium 1 x 10-3 NA 0.491 -

Mercury (methylmercury)d1 x 10-4 NA 0.049 -

Selenium 5 x 10-3 NA 2.457 -

Tributyltine3 x 10-4 NA 0.147 -

Organochlorine Pesticides

Total chlordane (sum of cis- and transchlordane, cis- and trans-nonachlor, and

oxychlordane)f

5 x 10-4 0.35 0.245 1.40 x 10-2

Total DDT (sum of 4,4'- and 2,4'- isomers

of DDT, DDE, and DDD)g

5 x 10-4 0.34 0.245 1.44 x 10-2

Dicofolh4 x 10-4 NAi0.196 -

Dieldrin 5 x 10-5 16 0.024 3.07 x 10-4

Endosulfan (I and II)j6 x 10-3 NA 2.949 -

Endrin 3 x 10-4 NA 0.147 -

Heptachlor epoxide 1.3 x 10-5 9.1 6.39 x 10-3 5.40 x 10-4

Hexachlorobenzene 8 x 10-4 1.6 0.393 3.07 x 10-3

Lindane (γ-hexachlorocyclohexane; γ-HCH)k 3 x 10-4 1.3 0.147 3.78 x 10-3

Mirex 2 x 10-4 NAl0.098 -

Toxaphenej,m 2.5 x 10-4 1.1 0.122 4.46 x 10-3

Organophosphate Pesticides

Chlorpyrifosn3 x 10-4 NA 0.147 -

Diazinono7 x 10-4 NA 0.344 -

Disulfoton 4 x 10-5 NA 0.019 -

Ethion 5 x 10-4 NA 0.245 -

Terbufosp2 x 10-5 NA 0.009 -

Chlorophenoxy Herbicides

Oxyfluorfenq3 x 10-3 7.32 x 10-2 1.474 6.71 x10-2

PAHsr NA 7.3 - 6.73 x 10-4

PCBs

Total PCBss2 x 10-5 2.0 9.83 x 10-3 2.45 x 10-3

Dioxins/furanst NA 1.56 x 105

- 3.15 x 10-8

NA = Not available in EPA’s Integrated Risk

Information System (IRIS, 1999).

DDD = p,p’-dichlorodiphenyldichloroethane

DDT = p,p’-dichlorodiphenyltrichloroethane

DDE = p,p’-dichlorodiphenlydichloroethylene

PAH = Polycyclic aromatic hydrocarbon

PCB = Polychlorinated biphenyl

RfD = Oral reference dose (mg/kg-d)

CSF = Cancer slope factor (mg/kg-d)-1
5. SCREENING VALUES FOR TARGET ANALYTES

5-14

Table 5-4. (continued)

a Based on fish consumption rate of 142.4 g/d, 70kg body weight and, for carcinogens, 10-5 risk level and 70-yr lifetime. Unless otherwise

noted, values listed are the most current oral RfDs and CSF in EPA’s IRIS database (IRIS, 1999)

b The shaded screening value (SV) is the recommended SV for each target analyte. States should note that the screening values listed may

be below analytical detection limits achievable for some of the target analytes. Please see Table 8-4 for detection limits.

c Total inorganic arsenic rather than total arsenic should be determined.

d Because most mercury in fish and shellfish tissue is present primarily as methylmercury (NAS, 1991;Tollefson, 1989) and because of the

relatively high cost of analyzing for methylmercury, it is recommended that total mercury be analyzed and the conservative assumption be

made that all mercury is present as methylmercury. This approach is deemed to be most protective of human health and most cost-effective.

The National Academy of Sciences conducted an independent assessment of the RfD for methylmercury. They concluded that “On the

basis of its evaluation, the committee’s consensus is that the value of EPA’s current RfD for methylmercury, 0.1Fg/kg per day, is a

scientifically justifiable level for the protection of human health”.

e The RfD value listed is for tributyltin oxide (IRIS, 1999).

f The RfD and CSF values listed are derived from studies using technical-grade chlordane (IRIS, 1999) for the cis- and trans-chlordane

isomers or the major chlordane metabolite, oxychlordane, or for the chlordane impurities cis- and trans-nonachlor. It is recommended that

total chlordane be determined by summing the concentrations of cis- and trans-chlordane, cis- and trans-nonachlor, and oxychlordane.

g The RfD value listed is for DDT. The CSF value (0.34) is for total DDT sum of DDT, DDE and DDD); the CSF value for DDD is 0.24. It

is recommended that the total concentration of DDT include the 2,4'- and 4,4'-isomers of DDT and its metabolites, DDE and DDD. 

h The RfD value is from Office of Pesticide Programs Reregistration Eligibility Decision (RED) for Dicofol (EPA, 1998c).

i The CSF for dicofol was withdrawn from IRIS pending further review by the CRAVE Agency Work Group (IRIS, 1999).

j The RfD value listed is from the Office of Pesticide Program’s Reference Dose Tracking Report (U.S. EPA, 1997).

k

IRIS (1999) has not provided a CSF for lindane. The CSF value listed for lindane was calculated from the water quality criteria (0.063 mg/L)

(U.S. EPA, 1992f).

l No CSF or cancer classification is available for mirex. This compound is undergoing further review by the CRAVE Agency Work Group

(IRIS, 1999)

m The RfD value has been agreed upon by the Office of Pesticide Programs and the Office of Water.

n Because of the potential for adverse neurological developmental effects from chlorpyrifos, EPA recommends the use of a Population

Adjusted Dose (PAD) of 3 x 10-5 for infants, children under the age of 6 years, and women ages 13 to 50 years (U.S. EPA, 2000b).

o The RfD value is from a memorandum dated April 1, 1998, Diazinon:-Report of the Hazard Identification Assessment Review Committee.

HED Doc. No. 012558.

p The RfD value listed is from a memorandum dated September 25, 1997; Terbufos-FQPA Requirement- Report of the Hazard Idenification

Review.

q The CSF value is from the Office of Pesticide Programs List of Chemicals Evaluated for Carcinogenic Potential (U.S. EPA, 1999b).

r The CSF value listed is for benzo[a]pyrene. Values for other PAHs are not currently available in IRIS (1999). It is recommended that tissue

samples be analyzed for benzo[a]pyrene and 14 other PAHs, and that the order-of-magnitude relative potencies given for these PAHs

(Nisbet and LaGoy, 1992; U.S. EPA, 1993c) be used to calculate a potency equivalency concentration (PEC) for each sample (see Section

5.3.2.4). 

s Total PCBs may be determined as the sum of congeners or Aroclors. The RfD is based on Aroclor 1254 and should be applied to total

PCBs. The CSF is based on a carcinogenicity assessment of Aroclors 1260, 1254, 1242, and 1016. The CSF presented is the upperbound slope factor for food chain exposure. The central estimate is 1.0 (IRIS, 1999). 

t The CSF value listed is for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (HEAST, 1997). It is recommended that the 17 2,3,7,8-substituted

tetra- through octa-chlorinated dibenzo-p-dioxins and dibenzofurans and the 12 dioxin-like PCBs be determined and a toxicity-weighted

total concentration be calculated for each sample, using the method for estimating toxicity equivalency concentrations (TEQs) (Van den

Berg et al., 1998). 
5. SCREENING VALUES FOR TARGET ANALYTES

5-15

the lower of the two SVs (generally, the SV for carcinogenic effects) should be

used for the respective fisher population. EPA recommends that the SVs in the

shaded boxes (Tables 5-3 and 5-4 ) be used by states when making the decision

to implement Tier 2 intensive monitoring. However, states may choose to adjust

these SVs for specific target analytes for the protection of sensitive populations

(e.g., pregnant women, nursing mothers, and children or for recreational or

subsistence fishers based on site-specific consumption rates). EPA recognizes

that states may use higher CRs that are more appropriate for recreational and

subsistence fishers in calculating SVs for use in their jurisdictions rather than the

EPA default values of 17.5 g/d CR for recreational fishers used to calculate the

SVs shown in Table 5-3 and the 142.4 g/d CR for subsistence fishers used to

calculate the SVs shown in Table 5-4. 

Note: States should use the same SV for a given target analyte in both

screening and intensive studies. Therefore, it is critical that states clearly define

their program objectives and accurately characterize the target fish-consuming

population(s) of concern to ensure that appropriate SVs are selected. If the

selected analytical methodology is not sensitive enough to reliably quantitate

target analytes at or below selected SVs (see Section 8.2.2 and Table 8-4),

program managers must determine appropriate fish consumption guidance based

on the lowest detectable concentrations or provide justification for adjusting SVs

to values at or above achievable method detection limits. It should be

emphasized that when SVs are below method detection limits, the failure to

detect a target analyte cannot be assumed to indicate that there is no cause for

concern for human health effects.

States should recognize the importance of ensuring that the analytical method

selected for quantification of any target analyte must have a method detection

limit (MDL) lower than the risk-based screening values calculated using the EPA

methodology for noncarcinogenic and carcinogenic effects of the target analyte.

If the method detection limit for a specific target analyte is higher than the target

analyte SV, the following procedure is recommended as a means to reduce the

problem of interpreting data results for chemicals that fall in this category. For

example, if fish tissue residue values for several replicate samples are above the

MDL while other data values are reported as below the method detection limit

(<MDL) including not detected (e.g., no observed response), the state may make

a risk management decision to use a value of one-half the MDL as the residue

concentration in their risk assessment for those data below the MDL rather than

using a value of zero. In this way, the calculated mean target analyte concentration for a group of replicate samples may be higher than the SV. If all of the

replicate samples from a particular monitoring site are below the MDL or are not

detected, the state may choose to use one-half MDL value for all not detected

values rather than a value of zero. The use of one-half MDL rather than zero for

these data (< MDL) is a risk management policy decision that should be made by

the state. 

For noncarcinogens, adjusted SVs should be calculated from Equation 5-4 using

appropriate alternative values of BW and/or CR. For carcinogens, adjusted SVs
5. SCREENING VALUES FOR TARGET ANALYTES

5-16

should be calculated from Equation 5-5 using an RL ranging from 10-4 to 10-7

and/or sufficiently protective alternative values of BW and CR. Examples of SVs

calculated for selected populations of concern and for RL values ranging from

10-4 to 10-7 are given in Table 5-5.

The need to accurately characterize the target fisher population of interest in

order to establish sufficiently protective SVs cannot be overemphasized. For

example, the recommended consumption rate of 142.4 g/d for subsistence

fishers may be an underestimate of consumption rate and exposures for some

subsistence populations such as Native American subsistence fishers (see

Section 5.1.3.2). In a recent study of a Native American subsistence fishing

population, an average daily consumption rate for these subsistence fishers was

estimated to be 540 g/d (Harris and Harper, 1997). Using this average

consumption rate and an estimated average body weight of 70 kg, the SV for

cadmium (RfD = 1 x 10-3 mg/kg/d) is, from Equation 5-4,

SV = (0.001 mg/kg-d • 70 kg) / (0.540 kg/d) = 0.129 mg/kg (ppm). (5-7)

This value is almost four times lower than the SV of 0.491 ppm for cadmium

based on the EPA default consumption rate of 142.4 g/d for subsistence fishers,

as shown in Table 5-4.

5.3 COMPARISON OF TARGET ANALYTE CONCENTRATIONS WITH SCREENING

VALUES

As noted previously, the same SV for a specific target analyte should be used in

both the screening and intensive studies. The measured concentrations of target

analytes in fish or shellfish tissue should be compared with their respective SVs

in both screening and intensive studies to determine the need for additional

monitoring and risk assessment.

Recommended procedures for comparing target analyte concentrations with SVs

are provided below. Related guidance on data analysis is given in Section 9.1.

5.3.1 Metals

5.3.1.1 Arsenic—

Most of the arsenic present in fish and shellfish tissue is organic arsenic, primarily

pentavalent arsenobetaine, which has been shown in numerous studies to be

metabolically inert and nontoxic (Brown et al., 1990; Cannon et al., 1983;

Charbonneau et al., 1978; Bos et al., 1985; Kaise et al. 1985; Luten et al., 1982;

Sabbioni et al., 1991; Siewicki, 1981; Bryce et al., 1982; Vahter et al., 1983;

Yamauchi et al., 1986). Inorganic arsenic, which is of concern for human health

effects (ATSDR, 1998a; WHO, 1989), is generally found in seafood at concentra-
5. SCREENING VALUES FOR TARGET ANALYTES

5-17

Table 5-5. Example Screening Values (SVs) for Various Target

Populations and Risk Levels (RLs)a

Chemical Target populationb CRc BW RfD CSF RL SV (ppm)

Noncarcinogens

Chlorpyrifos Recreational fisher 17.5 70 3 x 10-4 — — 1.2

Children (<6 yr) 6.5 17d 3 x 10-5e — — 0.078

Subsistence fisher 142.4 70 3 x 10-4 — — 0.147

Cadmium Recreational fisher 17.5 70 1 x 10-3 — — 4.0

Children 6.5 17d 1 x 10-3 — — 2.6

Subsistence fisher 142.4 70 1 x 10-3 — — 0.491

Carcinogens

Lindane Recreational fisher 17.5 70 — 1.3

1.3

1.3

1.3

10-4

10-5

10-6

10-7

3.07 x 10-1

3.07 x 10-2

3.07 x 10-3

3.07 x 10-4

Children 6.5 17d — 1.3

1.3

1.3

1.3

10-4

10-5

10-6

10-7

1.98 x 10-1

1.98 x 10-2

1.98 x 10-3

1.98 x 10-4

Subsistence fisher 142.4 70 — 1.3

1.3

1.3

1.3

10-4

10-5

10-6

10-7

3.78 x 10-2

3.78 x 10-3

3.78 x 10-4

3.78 x 10-5

Toxaphene Recreational fisher 17.5 70 — 1.1

1.1

1.1

1.1

10-4

10-5

10-6

10-7

3.63 x 10-1

3.63 x 10-2

3.63 x 10-3

3.63 x 10-4

Children 6.5 17d — 1.1

1.1

1.1

1.1

10-4

10-5

10-6

10-7

2.35 x 10-1

2.35 x 10-2

2.35 x 10-3

2.35 x 10-4

Subsistence fisher 142.5 70 — 1.1

1.1

1.1

1.1

10-4

10-5

10-6

10-7

4.6 x 10-2

4.6 x 10-3

4.6 x 10-4

4.6 x 10-5

CR = Mean daily fish or shellfish consumption rate (uncooked weight), averaged over a 70-yr lifetime for the

population of concern (g/d).

BW = Mean body weight, estimated for the population of concern (kg).

RfD = Oral reference dose for noncarcinogens (mg/kg-d).

CSF = Oral slope factor for carcinogens (mg/kg-d)-1.

RL = Maximum acceptable risk level for carcinogens (dimensionless).

a See Equations 5-4 and 5-5.

b See Tables 5-1, 5-2, 5-3 and 5-4 for information on target populations.

c To calculate SVs, the CRs given in this table must be divided by 1,000 to convert g/d to kg/d.

d BW used is for children 3 to <6 yr (see Table 5-1).

e Because of the potential for adverse neurological developmental effects, EPA recommends the use of a

Population Adjusted Dose for chlorpyrifos of 3 x 10-5 mg/kg-d for infants, children to the age of 6, and women

ages 13 to 50 years (U.S. EPA, 2000b).
5. SCREENING VALUES FOR TARGET ANALYTES

5-18

tions ranging from <1 to 20 percent of the total arsenic concentration (Edmonds

and Francesconi, 1993; Nraigu and Simmons, 1990). It is recommended that, in

both screening and intensive studies, total inorganic arsenic tissue

concentrations be determined for comparison with the recommended SV for

chronic oral exposure. This approach is more rigorous than the current FDArecommended method of analyzing for total arsenic and estimating inorganic

arsenic concentrations based on the assumption that 10 percent of the total

arsenic in fish tissue is in the inorganic form (U.S. FDA, 1993). Although the cost

of analysis for inorganic arsenic (see Table 8-5) may be three to five times

greater than for total arsenic, the increased cost is justified to ensure that the

most accurate data are obtained for quantitative assessment of human health

risks.

5.3.1.2 Cadmium, Mercury, and Selenium—

For cadmium, mercury, and selenium, the total metal tissue concentration should

be determined for comparison with the appropriate target population SV. 

Because most mercury in fish and shellfish tissue is present as methylmercury

(Kannan et al., 1998; NAS, 1991; Tollefson, 1989), and because of the relatively

high analytical cost for methylmercury, it is recommended that total mercury be

determined and the conservative assumption be made that all mercury is present

as methylmercury. The determination of methylmercury in fish tissue is not

recommended even though methylmercury is the compound of greatest concern

for human health (NAS, 1991; Tollefson, 1989) and the recommended SVs are

for methylmercury (see Tables 5-3 and 5-4). This approach is deemed to be

most protective of human health and most cost-effective.

5.3.1.3 Tributyltin—

Tissue samples should be analyzed specifically for tributyltin for comparison with

the recommended target population SVs for this compound (see Tables 5-3 and

5-4).

5.3.2 Organics

For each of the recommended organic target analytes that are single

compounds, the determination of tissue concentration and comparison with the

appropriate SV is straightforward. However, for those organic target analytes

that include a parent compound and structurally similar compounds or metabolites (i.e., total chlordane, total DDT, endosulfan I and II) or that represent classes

of compounds (i.e., PAHs, PCBs, dioxins/furans, or toxaphene), additional

guidance is necessary to ensure that a consistent approach is used to determine

appropriate target analyte concentrations for comparison with recommended

SVs.
5. SCREENING VALUES FOR TARGET ANALYTES

5-19

5.3.2.1 Chlordane—

The SVs for total chlordane are derived from technical-grade chlordane. Oral

cancer slope factors are not available in IRIS (1999) for cis- and trans-chlordane,

cis- and trans-nonachlor, and oxychlordane. At this time, as a conservative

approach, EPA recommends that, in both screening and intensive studies, the

concentrations of all chlordane constituents (cis- and trans-chlordane, cis- and

trans-nonachlor) and the metabolite of chlordane (oxychlordane) be determined

and summed to give a total chlordane concentration for comparison with the

recommended SVs (see Tables 5-3 and 5-4).

5.3.2.2 DDT—

DDT and its metabolites (i.e., the 4,4'- and 2,4'-isomers of DDE and DDD) are all

potent toxicants, DDE isomers being the most prevalent in the environment. As

a conservative approach, EPA recommends that, in both screening and intensive

studies, the concentrations of 4,4'- and 2,4'-DDT and their 4,4' and 2,4'-DDE and

DDD metabolites be determined and a total DDT concentration be calculated for

comparison with the recommended SVs for total DDT (see Tables 5-3 and 5-4).

5.3.2.3 Endosulfan—

Endosulfan collectively refers to two stereoisomers designated I and II. At this

time, for both screening and intensive studies, EPA recommends that the

concentrations of the two endosulfan constituents (endosulfan I and II) be

determined and summed to give a total endosulfan concentration for comparison

with the recommended SVs for total endosulfan.

5.3.2.4 Toxaphene—

The SVs for toxaphene are derived from technical-grade toxaphene, a mixture

of approximately 670 chlorinated camphenes (ATSDR, 1996). At this time,

determination of total toxaphene is recommended rather than individual congener

analysis. Research is currently under way to determine the relative health risks

of the toxaphene congeners. In the future, it may be possible to develop a

congener-specific quantitative risk assessment approach for toxaphene similar

to that for PCBs and dioxins/furans. The total toxaphene concentration should

be analyzed for comparison with the recommended SVs for toxaphene (see

Tables 5-3 and 5-4).

5.3.2.5 PAHs—

Although several PAHs have been classified as B2 carcinogens (probable human

carcinogens), benzo[a]pyrene is the only PAH for which a CSF is currently

available in IRIS (1999). As a result, EPA quantitative risk estimates for PAH

mixtures have often assumed that all carcinogenic PAHs are equipotent to

benzo[a]pyrene. The EPA Office of Health and Environmental Assessment has
5. SCREENING VALUES FOR TARGET ANALYTES

5-20

Table 5-6. Toxicity Equivalency Factors for Various PAHs

Compound Toxicity Equivalency Factor (TEF)

Dibenz[a,h]anthracene 5

Benzo[a]pyrene 1

Benz[a]anthracene 0

Benzo[b]fluoranthene 0.1

Benzo[k]fluoranthene 0.1

Indeno[1,2,3-cd]pyrene 0.1

Anthracene 0.01

Benzo[g,h,i]perylene 0.01

Chrysene 0.01

Acenaphthene 0.001

Acenaphthylene 0.001

Fluoranthene 0.001

Fluorene 0.001

Phenanthrene 0.001

Pyrene 0.001

Source: Nisbet and LaGoy (1992).

issued guidance for quantitative risk assessment of PAHs (Nisbet and LaGoy,

1992; U.S. EPA, 1993c) in which an estimated order of potential potency for

14 PAHs relative to benzo[a]pyrene is recommended, as shown in Table 5-6.

Based on this guidance, EPA recommends that, in both screening and intensive

studies, tissue samples be analyzed for the PAHs shown in Table 5-6 and that

a potency-weighted total concentration be calculated for each sample for

comparison with the recommended SVs for benzo[a]pyrene (see Tables 5-3 and

5-4). This potency equivalency concentration should be calculated using the

following equation:

PEC = 3

i

 (RPi · Ci) (5-8)

where

RPi = Relative potency for the ith PAH (from Table 5-6)

Ci = Concentration of the ith PAH.

5.3.2.6 PCBs—

Using the approach for PCB analysis recommended by the EPA Office of Water

(see Section 4.3.6), total PCB concentrations may be determined as the sum of

Aroclor equivalents in screening studies. For intensive studies, the total PCB

concentration should be determined as the sum of PCB congeners or the sum

of homologue groups. The total PCB concentration should be compared with the

recommended SVs for PCBs (see Tables 5-3 and 5-4). The EPA Office of Water

recognizes the potential problems associated with PCB congener analysis (i.e.,
5. SCREENING VALUES FOR TARGET ANALYTES

5-21

standard methods are not yet available but are under development, relatively

high analytical cost, and limited number of qualified laboratories), but is

recommending these methods for intensive studies because Aroclor analysis

does not adequately represent bioconcentrated PCB mixtures found in fish

tissue. EPA has developed a draft method for selected PCB congeners

(Method 1668) (U.S. EPA, 1997a). This method is being tested and may be

revised to include all PCB congeners. Currently, Method 680 is available for PCB

homologue analysis.

5.3.2.7 Dioxins and Dibenzofurans—

Note: At this time, EPA’s Office of Research and Development is reevaluating

the potency of dioxins/furans. Consequently, the following recommendation may

change pending the results of this reevaluation.

It is recommended in both screening and intensive studies that the 17 2,3,7,8-

substituted tetra- through octa-chlorinated PCDDs and PCDFs and the 12

coplanar congeners with dioxin-like effects be determined and that a toxicityweighted total concentration be calculated for each sample for comparison with

the recommended SVs for 2,3,7,8-TCDD (see Tables 5-3 and 5-4). 

The method for estimating total TEQ (Van den Berg et al., 1998) should be used

to estimate TCDD equivalent concentrations according to the following equation:

TEQ = 3

i

 (TEFi • Ci) (5-9)

where

TEFi = Toxicity equivalency factor for the ith congener (relative to 2,3,7,8-

TCDD)

Ci = Concentration of the ith congener.

TEFs for the 2,3,7,8-substituted tetra- through octa-PCDDs and PCDFs and the

12 dioxin-like PCBs are shown in Table 5-7. Note: TEFs for five congeners have

changed over those TEFs recommended by Barnes and Bellin (1989). 
5. SCREENING VALUES FOR TARGET ANALYTES

5-22

Table 5-7. Toxicity Equivalency Factors (TEFs) for Tetrathrough Octa-Chlorinated Dibenzo-p-Dioxins and Dibenzofurans

and Dioxin-Like PCBs

Analyte Old TEF-89 TEF-98

Dioxinsa

2,3,7,8-TCDD 1.00 1.00

1,2,3,7,8-PeCDD 0.50 1.00*

1,2,3,4,7,8-HxCDD

1,2,3,6,7,8-HxCDD

1,2,3,7,8,9-HxCDD

0.10 

0.10 

0.10 

0.10

0.10

0.10

1,2,3,4,6,7,8-HpCDD 0.01 0.01

OCDD 0.001 0.0001*

Furansa

2,3,7,8-TCDF 0.10 0.10

1,2,3,7,8-PeCDF

2,3,4,7,8-PeCDF

0.05 

0.50 

0.05

0.50

1,2,3,4,7,8-HxCDF

1,2,3,6,7,8-HxCDF

1,2,3,7,8,9-HxCDF

2,3,4,6,7,8-HxCDF

0.10 

0.10 

0.10 

0.10 

0.10

0.10

0.10

0.10

1,2,3,4,6,7,8-HpCDF

1,2,3,4,7,8,9-HpCDF

0.01 

0.01 

0.01

0.01

OCDF 0.001 0.0001*

PCBs

3,3',4,4'-TetraCB (77)

3,4,4',5-TetraCB (81)

0.0005

 not available

0.0001*

0.0001*

2,3,3',4,4'-PentaCB (105)

2,3,4,4',5-PentaCB (114)

2,3',4,4',5-PentaCB (118)

2',3,4,4',5-PentaCB (123)

3,3',4,4',5-PentaCB (126)

0.0001

0.0005

0.0001

0.0001

0.1

0.0001

0.0005

0.0001

0.0001

0.1

2,3,3',4,4',5-HexaCB (156)

2,3,3',4,4',5'-HexaCB (157)

2,3',4,4',5,5'-HexaCB (167)

3,3',4,4',5,5'-HexaCB (169)

2,3,3',4,4',5,5--HexaCB (189)

0.0005

0.0005

0.00001

0.01

0.0001

0.0005

0.0005

0.00001

0.01

0.0001

Sources: Barnes and Bellin, 1989; Van den Berg et al., 1998.

*Note: TEF-98 value changed from TEF-89 value.

aTEFs for all non-2,3,7,8-substituted congeners are zero.
6. FIELD PROCEDURES

6-1

SECTION 6

FIELD PROCEDURES

This section provides guidance on sampling design of screening and intensive

studies and recommends field procedures for collecting, preserving, and shipping

samples to a processing laboratory for target analyte analysis. Planning and

documentation of all field procedures are emphasized to ensure that collection

activities are cost-effective and that sample integrity is preserved during all field

activities. This section also describes the implications that result when deviations

occur in the recommended study design. Some of the deviations in study design

most likely to occur include the use of unequal numbers of fish in composite

samples, unequal numbers of replicate samples collected at different stations, and

sizes of fish within a composite sample exceeding the recommendation for

composite samples.

6.1 SAMPLING DESIGN

Prior to initiating a screening or intensive study, the program manager and field

sampling staff should develop a detailed sampling plan. As described in

Section 2, there are seven major parameters that must be specified prior to the

initiation of any field collection activities:

 Site selection

 Target species (and size class)

 Target analytes

 Target analyte screening values

 Sampling times

 Sample type

 Replicate samples.

In addition, personnel roles and responsibilities in all phases of the fish and

shellfish sampling effort should be defined clearly. All aspects of the final

sampling design for a state's fish and shellfish contaminant monitoring program

should be documented clearly by the program manager in a Work/QA Project

Plan (see Appendix I). Routine sample collection procedures should be prepared

as standard operating procedures (U.S. EPA, 1984b) to document the specific

methods used by the state and to facilitate assessment of final data quality and

comparability.

The seven major parameters of the sampling plan should be documented on a

sample request form prepared by the program manager for each sampling site.

The sample request form should provide the field collection team with readily

available information on the study objective, site location, site name/number,

target species and alternate species to be collected, target analytes to be

evaluated, anticipated sampling dates, sample type to be collected, number and
6. FIELD PROCEDURES

6-2

size range of individuals to be collected for each composite sample, sampling

method to be used, and number of replicates to be collected. An example of a

sample request form is shown in Figure 6-1. The original sample request form

should be filed with the program manager and a copy kept with the field logbook.

The seven major parameters that must be specified in the sampling plan for

screening and intensive studies are discussed in Sections 6.1.1 and 6.1.2,

respectively.

6.1.1 Screening Studies (Tier 1)

The primary aim of screening studies is to identify frequently fished sites where

commonly consumed fish and shellfish species are chemically contaminated and

may pose a risk to human health. Ideally, screening studies should include all

waterbodies where commercial, recreational, or subsistence fishing and shellfish

harvesting are practiced. 

6.1.1.1 Site Selection&

Sampling sites should be selected to identify extremes of the bioaccumulation

spectrum, ranging from presumed undisturbed reference sites to sites where

existing data (or the presence of potential pollutant sources) suggest significant

chemical contamination. Where resources are limited, states initially should target

those harvest sites suspected of having the highest levels of contamination and

of posing the greatest potential health risk to local fish and shellfish consumers.

Screening study sites should be located in frequently fished areas near

 Point source discharges such as

& Industrial or municipal discharges

& Combined sewer overflows (CSOs)

& Urban storm drains

 Nonpoint source inputs such as

& Landfills, Resource Conservation and Recovery Act (RCRA) sites, or

Superfund Comprehensive Environmental Response, Compensation, and

Liability Act (CERCLA) sites

& Areas of intensive agricultural, silvicultural, or resource extraction activities

or urban land development

& Areas receiving inputs through multimedia mechanisms such as

hydrogeologic connections or atmospheric deposition (e.g., areas affected

by acid rain impacts, particularly lakes with pH <6.0 since elevated

mercury concentrations in fish have been reported for such sites)

 Areas acting as potential pollutant sinks where contaminated sediments

accumulate and bioaccumulation potential might be enhanced (i.e., areas

where water velocity slows and organic-rich sediments are deposited)

 Areas where sediments are disturbed by dredging activities
6. FIELD PROCEDURES

6-3

Figure 6-1. Example of a sample request form.
6. FIELD PROCEDURES

6-4

 Unpolluted areas that can serve as reference sites for subsequent intensive

studies or as "green areas" that states can designate for unrestricted

consumption (see Appendix B). Note: Michigan sampled lakes that were in

presumed unpolluted areas but discovered mercury contamination in fish from

many of these areas and subsequently issued a fish consumption advisory for

all of its inland lakes.

The procedures required to identify candidate screening sites near significant

point source discharges are usually straightforward. It is often more difficult,

however, to identify clearly defined candidate sites in areas affected by pollutants

from nonpoint sources. For these sites, assessment information summarized in

state Section 305(b) reports should be reviewed before locations are selected.

State 305(b) reports are submitted to the EPA Assessment and Watershed

Protection Division biennially and provide an inventory of the water quality in each

state. The 305(b) reports often contain Section 319 nonpoint source assessment

information that may be useful in identifying major sources of nonpoint source

pollution to state waters. States may also use a method for targeting pesticide

hotspots in estuarine watersheds that employs pesticide use estimates from

NOAA's National Coastal Pollutant Discharge Inventory (Farrow et al., 1989).

It is important for states to identify and document at least a few unpolluted sites,

particularly for use as reference sites in subsequent monitoring studies.

Verification that targeted reference sites show acceptably low concentrations of

contaminants in fish or shellfish tissues also provides at least partial validation of

the methods used to select potentially contaminated sites. Clear differences

between the two types of sites support the site-selection methodology and the

assumptions about primary sources of pollution.

In addition to the intensity of subsistence, sport, or commercial fishing, factors that

should be evaluated (Versar, 1982) when selecting fish and shellfish sampling

sites include

 Proximity to water and sediment sampling sites

 Availability of data on fish or shellfish community structure

 Bottom condition

 Type of sampling equipment

 Accessibility of the site.

The most important benefit of locating fish or shellfish sampling sites near sites

selected for water and sediment sampling is the possibility of correlating

contaminant concentrations in different environmental compartments (water,

sediment, and fish). Selecting sampling sites in proximity to one another is also

more cost-effective in that it provides opportunities to combine sampling trips for

different matrices.

Availability of data on the indigenous fish and shellfish communities should be

considered in final site selection. Information on preferred feeding areas and
6. FIELD PROCEDURES

6-5

migration patterns is valuable in locating populations of the target species (Versar,

1982). Knowledge of habitat preference provided by fisheries biologists or

commercial fishermen may significantly reduce the time required to locate a

suitable population of the target species at a given site.

Bottom condition is another site-specific factor that is closely related to the

ecology of a target fish or shellfish population (Versar, 1982). For example, if only

soft-bottom areas are available at an estuarine site, neither oysters (Crassostrea

virginica) nor mussels (Mytilus edulis and M. californianus) would likely be present

because these species prefer hard substrates. Bottom condition also must be

considered in the selection and deployment of sampling equipment. Navigation

charts provide depth contours and the locations of large underwater obstacles in

coastal areas and larger navigable rivers. Sampling staff might also consult

commercial fishers familiar with the candidate site to identify areas where the

target species congregates and the appropriate sampling equipment to use.

Another factor closely linked to equipment selection is the accessibility of the

sampling site. For some small streams or land-locked lakes (particularly in

mountainous areas), it is often impractical to use a boat (Versar, 1982). In such

cases the sampling site should have good land access. If access to the site is by

land, consideration should be given to the type of vegetation and local topography

that could make transport of collection equipment difficult. If access to the

sampling site is by water, consideration should be given to the location of boat

ramps and marinas and the depth of water required to deploy the selected

sampling gear efficiently and to operate the boat safely. Sampling equipment and

use are discussed in detail in Section 6.2.1.

The selection of each sampling site must be based on the best professional

judgment of the field sampling staff. Once the site has been selected, it should

be plotted and numbered on the most accurate, up-to-date map available. Recent

7.5-minute (1:24,000 scale) maps from the U.S. Geologic Survey or blue line

maps produced by the U.S. Army Corps of Engineers are of sufficient detail and

accuracy for sample site mapping. The type of sampling to be conducted, water

depth, and estimated time to the sampling site from an access point should be

noted. The availability of landmarks for visual or range fixes should be

determined for each site, and biological trawl paths (or other sampling gear

transects) and navigational hazards should be indicated. Additional information

on site-positioning methods, including Loran-C, VIEWNAV, TRANSIT (NAVSAT),

GEOSTAR, and the NAVSTAR Global Positioning System (GPS), is provided in

Battelle (1986), Tetra Tech (1986), and Puget Sound Estuary Program (1990a).

Each sampling site must be described accurately because state fish and shellfish

contaminant monitoring data may be stored in a database available to users

nationwide (see Section 9.2). For example, a sampling site may be defined as a

2-mile section of river (e.g., 1 mile upstream and 1 mile downstream of a

reference point) or a 2-mile stretch of lake or estuarine/marine shoreline (U.S.

EPA, 1990d). Each sampler should provide a detailed description of each site
6. FIELD PROCEDURES

6-6

using a 7.5-minute USGS map to determine the exact latitude and longitude

coordinates for the reference point of the site. This information should be

documented on the sample request form and field record sheets (see

Section 6.2.3).

One additional consideration associated with sample site selection is whether the

sampling area includes waters inhabited by threatened or endangered species.

If such waterbodies are to be monitored, the state must obtain a permit from the

U.S. Fish and Wildlife Service (USFWS) if their sampling effort could potentially

impact a freshwater species (U.S. DOI, 1999) or from the National Marine

Fisheries Service (NMFS) if their sampling effort could potentially impact any

marine or anadromous species (U.S. DOC, 1999a, 1999b) covered under the

Endangered Species Act (ESA) of 1973. 

A species is listed under one of two categories, endangered or threatened,

depending on its status and the degree of threat it faces. An endangered species

is one that is in danger of extinction throughout all or a significant portion of its

range. A threatened species is one that is likely to become endangered in the

foreseeable future. The U.S. Fish and Wildlife Service maintains a list of all plant

and animal species native to the United States that are candidates or proposed

for possible addition to the Federal List. A complete listing of the current status

of all threatened and endangered species as well as information about each

USFWS region is available on-line on the USFWS website at

http://endangered.fws.gov/wildlife.html

Species information is also available by USFWS region having primary

responsibility for that species. The seven major USFWS regions with their

respective states are shown in Figure 6-2. States can obtain additional

information by contacting the specific USFWS regional office and talking with the

regional liaison for endangered species. 

Freshwater Threatened and Endangered Species

State conservation agencies typically have cooperative agreements in place with

the U.S. Fish and Wildlife Service. Under these agreements, any qualified

employee of the state agency may take those endangered species covered by the

cooperative agreement for conservation programs. Such taking of these species

may be done provided it does not result in the following:

 Death or permanent disabling of the specimen

 Rremoval of the specimen from the state where the taking occurred

 Introduction of the specimen so taken, or of any progeny derived from the

specimen, into an area beyond the historical range of the species

 Holding of the specimen in captivity for a period of more than 45 consecutive

days.
6. FIELD PROCEDURES

6-7

Additionally, any employee of a state conservation agency that is operating a

conservation program with the USFWS (in accordance with section 6(c) of the

Endangered Species Act) may take those threatened species of wildlife that are

covered by an approved cooperative agreement to carry out conservation

programs.

State agencies involved in designing and conducting fish sampling programs in

freshwater systems may need to sample fish for human health risk assessments

from areas inhabited by threatened or endangered species. In some of these

waterbodies under study, threatened or endangered species may be collected

incidental to the primary sampling objective. In these cases, the state agency

involved in the primary sampling needs to check with the state conservation

agency to determine whether a cooperative agreement between the state and the

USFWS is in effect. Any questions about the permits for incidental taking of

endangered or threatened species resulting from fish sampling programs should

be reviewed with the appropriate USFWS regional endangered species liaison

officer. If appropriate, the state must apply to the USFWS for an Incidental Take

Permit (U.S. DOI, 1999). States are required to submit information on USFWS

Form 3-200 with all of the following information provided as part of the permit

application:

Figure 6-2. U.S. Fish and Wildlife Service Regions.
6. FIELD PROCEDURES

6-8

 A complete description of the sampling activity sought to be authorized

 The common and scientific names of the species sought to be covered by the

permit, as well as the number, age, and sex of such species, if known.

The application must also include a conservation plan that specifies

 The impact that will likely result from such incidental taking

 What steps the applicant will take to monitor, minimize, and mitigate such

impacts, the funding that will be available to implement such steps, and the

procedures to be used to deal with unforseen circumstances

 What alternative actions to such incidental taking the applicant considered and

the reasons why such alternatives are not proposed to be used

 Such other measures that the Director may require as being necessary or

appropriate for purposes of the plan.

The completed application should be submitted to

U.S. Fish and Wildlife Service

Ecological Services/Endangered Species Permits

Attention: Regional Permit Coordinator

(see addresses below for each of the seven USFWS regional offices)

Region 1

Pacific Region

Eastside Federal Complex

911 NE 11th Avenue

Portland, OR 97232-4181

Region 2

Southwest Region

P.O. Box 1306

Albuquerque, NM 87103-1306

Region 3

Great Lakes and Big Rivers Region

1 Federal Drive

BHW Federal Building

Fort Snelling, MN 55111

Region 4

Southeast Region

1875 Century Boulevard, Suite 400

Atlanta, GA 30345-3319

Region 5

Northeast Region

300 Westgate Center Drive

Hadley, MA 01035-9589

Region 6

Mountain Prairie Region

134 Union Boulevard

Lakewood, CO 80228 

Region 7

Alaska Region

300 Vintage Boulevard, Suite 201

Juneau, AK 99801-7125
6. FIELD PROCEDURES

6-9

States should expect to wait from 3 to 6 months to obtain such a permit and

should plan and schedule their permit application submission accordingly. 

Marine or Anadromous Threatened and Endangered Species 

Each state that intends to sample fish as part of their tissue residue monitoring

program and might collect endangered or threatened marine or anadromous

species incidental to the purpose of their monitoring effort, must apply to the

NMFS for an Incidental Take Permit (U.S. DOC, 1999a). Application forms and

detailed instructions for completing these permit applications are available for

downloading on the Internet at url:

http://www.nmfs.noaa.gov/prot_res/PR3/Permits/ESAPermit.html. Users

should click on <<Incident Take of Listed Species>> under Activity Category and

select the PDF or HTML instructions. 

States are required to submit information about the following:

 Type of permit

 Date of application

 Name, address, telephone, and fax number of the applicant

 A description of the endangered or threatened species, by common and

scientific name, and a description of the status distribution, seasonal

distribution, habitat needs, feeding habits, and other biological requirements

of the affected species

 A detailed description of the proposed sampling activity, including

& Anticipated dates and duration of sampling activity

& Specific location of the activity (latitude and longitude coordinates)

& An estimate of the total level of activity expected to be conducted

The application must also include a conservation plan based on the best scientific

and commercial data available, which specifies

 Anticipated impact of the proposed activity on the listed species, including

& Estimated number of animals of the listed species and, if applicable, the

subspecies or population group and range

& Type of anticipated taking, such as harassment, predation, competition for

space and food, etc.

& Effects of the take on the listed species, such as descaling, altered

spawning activities, potential for mortality
6. FIELD PROCEDURES

6-10

 Anticipated impact of the proposed activity on the habitat of the species and

the likelihood of restoration of the affected habitat

 Steps that will be taken to monitor, minimize, and mitigate such impacts,

including

& Specialized equipment, methods of conducting activities, or other means.

& Detailed monitoring plans

& Funding available to implement measures taken to monitor, minimize, and

mitigate impacts.

 Alternative actions to such taking that were considered and the reasons why

those alternatives are not being used.

 A list of all sources of data used in preparation of the plan, including reference

reports, environmental assessments and impact statements, and personal

communications with recognized experts on the species or activity who may

have access to data not published in the current literature.

The application may be submitted electronically if possible (either by e-mail or by

mailing a diskette), but one signed original of the complete application must be

sent to

Chief, Endangered Species Division

National Marine Fisheries Service, F/PR3

1315 East-West Highway

Silver Spring, Maryland 20910

Telephone (301) 713-1401, Fax (301) 713-0376

States should expect to wait from 3 to 6 months to obtain such a permit and

should plan and schedule their permit application submission accordingly. 

Threatened or Endangered Sea Turtles

States planning on sampling fish in marine waters inhabited by threatened or

endangered species of sea turtles must apply to the NMFS for a Sea Turtle

Incidental Take Permit (U.S. DOC, 1999b). 

Application forms and detailed instructions for completing these permit

applications are available for downloading on the Internet at

http://www.nmfs.noaa.gov/prot_res/PR3/Permits/ESAPermit.html.

States are required to submit a cover letter including information on the following:

 Type of permit

 Date of application
6. FIELD PROCEDURES

6-11

 Name, address, telephone, and fax number of the applicant

 A description of each endangered or threatened sea turtle species impacted

by the activity, by common and scientific name, and a description of the

status, geographic distribution, seasonal distribution, habitat needs, feeding

habits, and other biological requirements of the affected species

 A detailed description of the proposed sampling activity (fishery season),

including

& Anticipated dates and duration of sampling activity

& Specific location of the activity (latitude and longitude coordinates) and

fishery effort in that area

& Other relevant information (e.g., gear description.)

The application must also submit a Conservation Plan based on the best scientific

and commercial data available. The Conservation Plan must emphasize techniques, gear types, and general practices to mitigate takes. The Conservation

Plan may involve development of new gear types or modification of fishing

practices and include the following information

 Anticipated impact of the activity on the listed species of sea turtle, including

& Estimated number of animals of the listed species impacted, their

geographic range, and, if applicable, the subspecies or population group,

& Type of anticipated taking, such as capture, harassment, predation,

competition for space and food, nature of injury

& Effects of the impact on the listed species, such as descaling, altered

reproductive activities, potential for mortality, effects of repeated

submergence

 Anticipated impact of the proposed activity on the habitat of the species and

the likelihood of restoration of the affected habitat

 Steps that will be taken to monitor, minimize, and mitigate such impacts,

including

& Detailed monitoring plans (e.g., observer programs)

& Detailed enforcement plans (e.g., monitoring Turtle Excluder Device

compliance)

& Specialized equipment, methods of conducting activities, or other

mitigation techniques.

& Detailed funding plan to implement measures taken to monitor, minimize,

and mitigate impacts.

 Alternatives to the activity considered and the reasons why those alternatives

are not being used.
6. FIELD PROCEDURES

6-12

 A list of all sources of data used in preparation of the plan, including reference

reports, environmental assessments and impact statements, and personal

communications with recognized experts on the species or activity who may

have access to data not published in the current literature.

 Other measures the Assistant Administrator of NMFS may require as

necessary or appropriate for the purposes of the plan.

The following criteria are considered for permit issuance:

 Status of the stock and/or species to be incidentally taken

 Likely direct and indirect impacts of the activity on sea turtles

 Availability and effectiveness of monitoring and enforcement programs

 Public comments received during the 30-day public notice and comment

period

 Adequate funding for the Conservation Plan

 The fact that taking will not appreciably reduce the likelihood of survival and

recovery of the species in the wild. 

An issued permit would

 Require regular reporting and rights of inspection

 Identify species and number of animals allowed to be taken incidentally

 Specify the authorized method of incidental taking

 Require procedures for captured sea turtles (i.e., resuscitation techniques,

disposal)

 Potentially impose administrative fees

 Establish duration of the permit

 Specify any other terms or conditions that the Assistant Administrator of

NMFS identifies as necessary or appropriate

 The application may be submitted electronically if possible (either by e-mail

or by mailing a diskette), but one signed original of the complete application

must be sent to
6. FIELD PROCEDURES

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Chief, Endangered Species Division

National Marine Fisheries Service, F/PR

1315 East-West Highway

Silver Spring, Maryland 20910

Telephone (301) 713-1401, Fax (301) 713-0376

States should expect to wait from 3 to 6 months to obtain such a permit and

should plan and schedule their permit application submission accordingly. 

6.1.1.2 Target Species and Size Class Selection&

After reviewing information on each sampling site, the field collection staff should

identify the target species that are likely to be found at the site. Target species

recommended for screening studies in freshwater systems are shown in

Tables 3-1, 3-2, and 3-4. Tables 3-10 through 3-16 list recommended species for

estuarine/marine areas. In freshwater ecosystems, one bottom-feeding and one

predator fish species should be collected. In estuarine/marine ecosystems, either

one bivalve species and one finfish species or two finfish species should be

collected. Second- and third-choice target species should be selected in the

event that the recommended target species are not collected at the site. The

same criteria used to select the recommended target species (Section 3.2) should

be used to select alternate target species. In all cases, the primary selection

criterion should be that the target species is commonly consumed locally and is

of harvestable size.

EPA recognizes that resource limitations may influence the sampling strategy

selected by a state. If monitoring resources are severely limited, precluding

performance of any Tier 2 intensive studies (Phase I and Phase II), EPA

recommends three sampling options to states for collecting additional samples

during the screening studies. These options are:

1. Collecting one composite sample for each of three size (age) classes of each

target species

2. Collecting replicate composite samples for each target species

3. Collecting replicate composite samples for each of three size (age) classes of

each target species.

Option 1 (single composite analysis for each of three size classes) provides

additional information on size-specific levels of contamination that may allow

states to issue an advisory for only the most contaminated size classes while

allowing other size classes of the target species to remain open to fishing. The

state could analyze the composite sample from the largest size class first. If any

SVs are exceeded, analysis of the smaller size class composite samples could be

conducted. This option, however, does not provide any additional information for

estimating the variability of the contamination level in any specific size class. To

obtain information for estimating the variability of the contamination level in the

target species, states could separately analyze each individual fish specimen in
6. FIELD PROCEDURES

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any composite that exceeded the SVs. Note: This option of analyzing individual

fish within a composite sample is more resource-intensive with respect to

analytical costs but is currently used by some Great Lakes states.

Option 2 (replicate analyses of one size class) provides additional statistical power

that would allow states to estimate the variability of contamination levels within the

one size class sampled; however, it does not provide information on size-specific

contamination levels.

Option 3 (replicate analyses of three size classes) provides both additional

information on size-specific contamination levels and additional statistical power

to estimate the variability of the contaminant concentrations in each of three size

classes of the target species. If resources are limited, the state could analyze the

replicate samples for the largest size class first; if the SVs are exceeded, analysis

of the smaller size class composite samples could then be conducted.

Note: The correlation between increasing size (age) and contaminant tissue

concentration observed for some freshwater finfish species (Voiland et al., 1991)

may be much less evident in estuarine/marine finfish species (G. Pollock,

California Environmental Protection Agency, personal communication, 1993). The

movement of estuarine and marine species from one niche to another as they

mature may change their exposure at a contaminated site. Thus, size-based

sampling in estuarine/marine systems should be conducted only when it is likely

to serve a potential risk management outcome.

6.1.1.3 Target Analyte Selection&

All 25 recommended target analytes listed in Table 4-1 should be considered for

inclusion in screening studies unless reliable historic tissue, sediment, or pollutant

source data indicate that an analyte is not present at a level of concern for human

health. Additional regional or site-specific target analytes should be included in

screening studies when there is indication or concern that such contaminants are

a potential health risk to local fish or shellfish consumers. Historic data on water,

sediment, and tissue contamination and priority pollutant scans from known point

source discharges or nonpoint source monitoring should be reviewed to determine

whether analysis of additional analytes is warranted.

6.1.1.4 Target Analyte Screening Values&

To enhance national consistency in screening study data, states should use the

target analyte screening values listed in Tables 5-3 and 5-4 to evaluate tissue

contaminant data. Specific methods used to calculate SVs for noncarcinogenic

and carcinogenic target analytes, including examples of SVs calculated for

selected subpopulations, are given in Sections 5.1 and 5.2. If target analytes

different from those default SVs shown in Tables 5-3 and 5-4 are included in a

screening study, these calculation procedures should be used to estimate SVs

based on typical exposure assumptions for the fish-consuming public for the
6. FIELD PROCEDURES

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additional compounds. Note: If the state chooses to use a different risk level or

consumption rate to address site-specific considerations, the corresponding SVs

should be calculated prior to initiation of chemical analyses to ensure that the

detection limits of the analytical procedures are sufficiently low to allow reliable

quantitation at or below the chosen SV. If analytical methodology is not sensitive

enough to reliably quantitate target analytes at or below selected SVs (see

Sections 5.2 and 8.2.2 and Table 8-4), program managers must determine

appropriate fish consumption guidance based on lowest detectable concentrations

or provide justification for adjusting SVs to values at or above achievable method

detection limits. It should be emphasized that when SVs are below method

detection limits, the failure to detect a target analyte cannot be assumed to

indicate that there is no cause for concern for human health effects.

6.1.1.5 Sampling Times&

If program resources are sufficient, biennial screening of waterbodies is recommended where commercial, recreational, or subsistence harvesting is commonly

practiced (as identified by the state). Data from these screenings can then be

used in the biennial state 305(b) reports to document the extent of support of

Clean Water Act goals. If biennial screening is not possible, then waterbodies

should be screened at least once every 5 years.

Selection of the most appropriate sampling period is very important, particularly

when screening studies may be conducted only once every 2 to 5 years. Note:

For screening studies, sampling should be conducted during the period when the

target species is most frequently harvested (U.S. EPA, 1989d; Versar, 1982).

In fresh waters, as a general rule, the most desirable sampling period is from late

summer to early fall (i.e., August to October) (Phillips, 1980; Versar, 1982). The

lipid content of many species (which represents an important reservoir for organic

pollutants) is generally highest at this time. Also, water levels are typically lower

during this time, thus simplifying collection procedures. This late summer to early

fall sampling period should not be used, however, if (1) it does not coincide with

the legal harvest season of the target species or (2) the target species spawns

during this period. Note: If the target species can be legally harvested during its

spawning period, however, then sampling to determine contaminant

concentrations should be conducted during this time.

A third exception to the late summer to early fall sampling recommendation

concerns monitoring for the organophosphate pesticides. Sampling for these

compounds should be conducted during late spring or early summer within 1 to

2 months following pesticide application because these compounds are degraded

and metabolized relatively rapidly compared to organochlorine pesticides. Note:

The target species should be sampled during the spring only if the species can be

legally harvested at this time.
6. FIELD PROCEDURES

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In estuarine and coastal waters, the most appropriate sampling time is during the

period when most fish are caught and consumed (usually summer for recreational

and subsistence fishers). For estuarine/marine shellfish (bivalve molluscs and

crustaceans), two situations may exist. The legal harvesting season may be

strictly controlled for fisheries resource management purposes or harvesting may

be open year round. In the first situation, shellfish contaminant monitoring should

be conducted during the legal harvest period. In the second situation, monitoring

should be conducted to correspond to the period when the majority of harvesting

is conducted during the legal season. state staff may have to consider different

sampling times for target shellfish species if differences in the commercial and

recreational harvesting period exist.

Ideally, the sampling period selected should avoid the spawning period of the

target species, including the period 1 month before and 1 month after spawning,

because many aquatic species are subject to stress during spawning. Tissue

samples collected during this period may not always be representative of the

normal population. For example, feeding habits, body fat (lipid) content, and

respiration rates may change during spawning and may influence pollutant uptake

and clearance. Collecting may also adversely affect some species, such as trout

or bass, by damaging the spawning grounds. Most fishing regulations protect

spawning periods to enhance propagation of important fishery species. Speciesspecific information on spawning periods and other life history factors is available

in numerous sources (e.g., Carlander, 1969; Emmett et al., 1991; Pflieger, 1975;

Phillips, 1980). In addition, digitized life history information is available in many

states through the Multistate Fish and Wildlife Information Systems (1990) on the

web at http://fwie.fw.vt.edu.

Exceptions to the recommended sampling periods for freshwater and estuarine/

marine habitats will be determined by important climatic, regional, or site-specific

factors that favor alternative sampling periods. For many states, budgetary

constraints may require that most sampling be conducted during June, July, and

August when temporary help or student interns are available for hire. The actual

sampling period and the rationale for its selection should be documented fully and

the final data report should include an assessment of sampling period effects on

the results.

6.1.1.6 Sample Type&

Composite samples of fish fillets or of the edible portions of shellfish are

recommended for analysis of target analytes in screening studies (U.S. EPA

1987b; 1989d). For health risk assessments, the recommended composite

sample type for chemical analysis should be based on both the study objectives

and the sample type consumed by the target population of concern. For

example, using skinless fillets for assessing mercury exposures for members of

the general population and most recreational fishers is most conservative.

Because mercury is differentially concentrated in muscle tissue, leaving the skin

on the fish fillet actually results in a lower mercury concentration per gram of skin-
6. FIELD PROCEDURES

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on fillet than per gram of skin-off fillet (Gutenmann and Lisk, 1991). In addition,

few consumers in the general population eat the skin of the fish, which justifies

its removal for analysis, particularly when monitoring concerns are directed solely

at mercury contamination. Analysis of skinless fillets may also be more

appropriate for some target species such as catfish and other scaleless finfish

species. In contrast, using whole fish with skin-on as the sample type for

assessing PCBs, dioxins/furans, or organochlorine pesticide exposures in

populations of Native Americans, Asian Americans, Caribbean-Americans, or

other ethnic groups that consume whole fish in a stew or soup is warranted

because these contaminants accumulate in fatty tissues of the fish. Cooking the

whole fish to make a stew or soup releases the PCBs, dioxins/furans, or

organochlorine contaminants into the broth; thus, the whole fish should be

analyzed to mirror the way the consumer prepares the fish. Similarly, using skinon fillets with belly-flap included for most other scaled fish to evaluate PCB,

dioxin/furan, or organochlorine pesticide exposures in the general fishing population or among recreational fishers is appropriate since this is a standard filleting

method (see Sections 7.2.2.6 and 7.2.2.7). This method also allows for the

inclusion of the fatty belly flap tissue and skin in which organochlorines, PCBs,

and dioxins/furans concentrate and takes into account the fact that some

consumers may not neatly trim the more highly contaminated fatty tissue from the

edible muscle fillet tissue. 

For shellfish samples, the recommended composite sample type for chemical

analysis also should be based on both the study objectives and the sample type

consumed by the target population at risk. The specific tissues considered to be

edible will vary among target shellfish species (see Section 7.2.4.4) based on

local consumer preference. For example, several states (Maine, Massachusetts,

New Hampshire, New Jersey and New York) have issued advisories for a variety

of contaminants (PCBs, dioxins/furans, or cadmium) in specific glands or tissues

of crustaceans such as lobsters and crabs. Some consumers of lobsters,

Homarus americanus, enjoy eating the tomalley (digestive gland of the lobster),

which has been shown to contain higher concentrations of chemical contaminants

than the claw, leg, or tail meat typically consumed by members of the general

population. For this reason, the tomalley should be analyzed separately if the

target population consumes this organ so that a determination can be made as

to whether contaminant concentrations in the tomalley only, or in the claw, leg,

and tail meat are above levels of human health concern. Similarly, for the blue

crab, Callinectes sapidus, as well as other crab species, the hepatopancreas

(digestive gland) is consumed by some individuals and has also been found to

contain higher concentrations of contaminants than claw, leg, or body muscle

tissue. If the target population of concern consumes the hepatopancreas, then

to best evaluate the risk of consumption from this tissue, it should be analyzed

separately from the claw, leg, and body muscle tissue. A precise description of

the sample type (including the number and size of the individual crustaceans in

the composite) should be documented in the program record for each target

species.
6. FIELD PROCEDURES

6-18

A similar situation exists with respect to selection of the appropriate sample type

for bivalve molluscs. For example, while most individuals in the general population consume whole oysters (e.g.,Crassostrea virginica or C. gigas), clams (e.g.,

Mercenaria mercenaria) or mussels (e.g., Mytilus edulis or M. californianus), only

the adductor muscle tissue is typically consumed of the scallops (Aropecten

irradians or A. gibbus). For bivalves in general, the adductor muscle is typically

less contaminated than gill, mantle, and digestive organ tissues primarily due to

the filter-feeding nature of these animals. Therefore, the adductor muscle of

scallops should be analyzed separately for the general population. If the whole

body of the scallop is to be consumed as part of a stew or soup by the target

population of concern, the state should also conduct analysis of the whole body

of the scallop as part of a risk assessment. A precise description of the sample

type (including the number and size of the individual bivalves in the composite)

should be documented in the program record for each target species.

For freshwater turtles also, the study objectives and sample type consumed by

the target population at risk must be of primary consideration. However, EPA

recommends use of individual turtle samples rather than composite samples for

evaluating turtle tissue contamination. As with shellfish, the tissues of freshwater

turtles considered to be edible vary based on the dietary and culinary practices

of local populations (see Section 7.2.3.3). For example, New York and Minnesota

have advisories for snapping turtles that recommend that consumers who wish to

eat turtle meat should trim away all fat and discard the liver and eggs of the turtle

(if they are still in the female’s body cavity) prior to cooking. These three tissues

(fat, liver, and eggs) have been shown to accumulate extremely high concentrations of a variety of contaminants in comparison to muscle tissue (Bishop et al.,

1996; Bonin et al. 1995; Bryan et al., 1987; Hebert et al., 1993; Olafsson et al.,

1983; 1987; Ryan et al., 1986; and Stone et al., 1980). States should consider

monitoring pollutant concentrations in all three tissues in addition to muscle tissue.

If residue analysis reveals the presence of high concentrations of contaminants

in liver, eggs, and fatty tissue, but not in the muscle tissue, then the state can

make the general recommendation to consumers to discard the three most

lipophilic tissues to reduce the risk of exposure. This action is most useful when

such lipophilic contaminants such as dioxins/furans, PCBs, and organochlorine

pesticides are the contaminants involved. 

Note: Composite samples are homogeneous mixtures of samples from two or

more individual organisms of the same species collected at a particular site and

analyzed as a single sample. Because the costs of performing individual chemical

analyses are usually higher than the costs of sample collection and preparation,

composite samples are most cost-effective for estimating average tissue

concentrations of target analytes in target species populations. Besides being

cost-effective, composite samples also ensure adequate sample mass to allow

analyses for all recommended target analytes. A disadvantage of using

composite samples, however, is that extreme contaminant concentration values

for individual organisms are lost.
6. FIELD PROCEDURES

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In screening studies, EPA recommends that states analyze one composite

sample for each of two target species at each screening site. Organisms used in

a composite sample

 Must all be of the same species

 Should satisfy any legal requirements of harvestable size or weight, or at least

be of consumable size if no legal harvest requirements are in effect

 Should be of similar size so that the smallest individual in a composite is no

less than 75 percent of the total length (size) of the largest individual

 Should be collected at the same time (i.e., collected as close to the same time

as possible but no more than 1 week apart) [Note: This assumes that a

sampling crew was unable to collect all fish needed to prepare the composite

sample on the same day. If organisms used in the same composite are

collected on different days (no more than 1 week apart), they should be

processed within 24 hours as described in Section 7.2 except that individual

fish may have to be filleted and frozen until all the fish to be included in the

composite are delivered to the laboratory. At that time, the composite

homogenate sample may be prepared.]

 Should be collected in sufficient numbers to provide a 200-g composite

homogenate sample of edible tissue for analysis of recommended target

analytes.

Individual organisms used in composite samples must be of the same species

because of the significant species-specific bioaccumulation potential. Accurate

taxonomic identification is essential in preventing the mixing of closely related

species with the target species. Note: Individuals from different species should

not be used in a single composite sample (U.S. EPA, 1989d, 1990d).

For cost-effectiveness, EPA recommends that states collect only one size class

for each target species and focus on the larger individuals commonly harvested

by the local population. Ideally, each composite sample for a specific species

should contain the same number of individual fish and the individuals within each

target species composite should be of similar size within a target size range so

that the composite samples for a particular species are comparable over a wide

geographic area. This is particularly important when states want to compare data

on an individual species that might be used to establish a statewide advisory. 

For persistent chlorinated organic compounds (e.g., DDT, dioxin, PCBs, and

toxaphene) and methylmercury, the larger (older) individuals within a population

are generally the most contaminated (Phillips, 1980; Voiland et al., 1991). As

noted earlier, this correlation between increasing size and increasing contaminant

concentration is most striking in freshwater finfish species but is less evident in

estuarine and marine species. Size is used as a surrogate for age, which
6. FIELD PROCEDURES

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provides some estimate of the total time the individual organism has been at risk

of exposure. Therefore, the primary target size range ideally should include the

larger individuals harvested at each sampling site. In this way, the states will

maximize their chances of detecting high levels of chemical contamination in the

single composite sample collected for each target species. If this ideal condition

cannot be met, the field sampling team should retain individuals of similar length

that fall within a secondary target size range.

Individual organisms used in composite samples should be of similar size (WDNR,

1988). Note: Ideally, for fish or shellfish, the total length (or size) of the smallest

individual in any composite sample should be no less than 75 percent of the total

length (or size) of the largest individual in the composite sample (U.S. EPA,

1990d). For example, if the largest fish is 200 mm, then the smallest individual

included in the composite sample should be at least 150 mm. In the California

Mussel Watch Program, a predetermined size range (55 to 65 mm) for the target

bivalves (Mytilus californianus and M. edulis) is used as a sample selection

criterion at all sampling sites to reduce size-related variability (Phillips, 1988).

Similarly, the Texas Water Commission (1990) specifies the target size range for

each of the recommended target fish species collected in the state's fish

contaminant monitoring program.

Individual organisms used in a composite sample ideally should be collected at

the same time so that temporal changes in contaminant concentrations

associated with the reproduction cycle of the target species are minimized.

Each composite sample should contain 200 g of tissue so that sufficient material

will be available for the analysis of all recommended target analytes. A larger

composite sample mass may be required when the number of target analytes is

increased to address regional or site-specific concerns. However, the tissue

mass may be reduced in the Tier 2 intensive studies (Phase I and II) when a

limited number of specific analytes of concern have been identified (see Section

7.2.2.9). Given the variability in size among target species, only approximate

ranges can be suggested for the number of individual organisms to collect to

achieve adequate mass in screening studies (U.S. EPA, 1989d; Versar, 1982).

For fish, 3 to 10 individuals should be collected for a composite sample for each

target species; for shellfish, 3 to 50 individuals should be collected for a composite

sample. In some cases, however, more than 50 small shellfish (e.g., mussels,

shrimp, crayfish) may be needed to obtain the recommended 200-g sample mass.

Note: The same number of individuals should be used in each composite sample

for a given target species at each sampling site.

Deviations from the recommended study design have implications that may make

the statistical analyses more complicated. The statistical methods for analyzing

composite samples are made tractable and easier-to-use by simplifying the study

design. Using equal numbers of fish in replicate composite samples is one way

to do this. For example, with equal numbers of fish, the arithmetic average of the

replicate composite measurements is an unbiased estimator of the population
6. FIELD PROCEDURES

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mean. When unequal numbers are used, the arithmetic average is no longer

unbiased. Instead, a weighted average of the composite measurements is

calculated, where the weight for each composite reflects the number of fish it is

made up of. Oftentimes fish are lost or damaged prior to compositing. When

several fish are damaged or lost, the allocation of the remaining fish to

composites may be reconfigured to allow equal numbers of fish in composites. If

this is not possible, care should be taken to adjust the statistical procedures to

account for the unequal allocations.

The use of sizes of fish exceeding the size range recommended for compositing

may introduce more variability. If it is the size range within each composite that

is broadened (e.g., 100-200 mm instead of 150-200 mm), the variability within the

composite may increase. If additional composites are made with fish exceeding

the recommended size ranges (e.g., adding composites of fish of size 300-450

mm when the target size is no more than 250 mm), this may increase the

variability between composites of different size ranges. Overall inferences made

from composites of different size ranges will have increased variability associated

with them (e.g., wider confidence intervals).

Differences in the numbers of replicates at different sampling locations may

complicate any comparisons to be made between locations or overall conclusions

to be obtained by combining the results from different sampling locations. As with

unequal numbers of fish in composites, unequal numbers of replicate samples

complicate the statistical calculations. The appropriate weighted estimates should

be used when combining information from different sampling locations. Consider,

for instance, a state that monitors five lakes each year. If the state uses the same

target fish species, the same number of fish per composite and the same size

ranges, the overall mean level of contamination will be a straightforward average

over the five locations if the same number of replicates are used at each location.

However, if unequal numbers of replicates are used, the information contributed

by each location is not the same and must be weighted accordingly.

As alluded to above, one limitation of using composite samples is that information

on extreme levels of chemical contamination in individual organisms is lost.

Therefore, EPA recommends that the residual individual homogenates be saved

to allow for analyses of individual specimens if resources permit (Versar, 1982).

Analysis of individual homogenates allows states to estimate the underlying

population variance which, as described in Section 6.1.2.6, facilitates sample size

determination for the intensive studies. Furthermore, individual homogenates

may also be used to provide materials for split and spike samples for routine QC

procedures either for composites or individual organisms (see Section 8.3). The

circumstances in which the analysis of individual fish samples might be preferred

over the analysis of composite samples is described in more detail in Appendix C.

Recommended sample preparation procedures are discussed in Section 7.2.
6. FIELD PROCEDURES

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6.1.1.7 Replicate Samples&

The collection of sufficient numbers of individual organisms from a target species

at a site to allow for the independent preparation of more than one composite

sample (i.e., sample replicates) is strongly encouraged but is option in screening

studies. If resources and storage are available, single replicate (i.e., duplicate)

composite samples should be collected at a minimum of 10 percent of the

screening sites (U.S. EPA, 1990d). The collection and storage of replicate

samples, even if not analyzed at the time due to inadequate resources, allow for

followup QC checks. These sites should be identified during the planning phase

and sample replication specifications noted on the sample request form. If

replicate field samples are to be collected, states should follow the guidance

provided in Section 6.1.2.7. Note: Additional replicates must be collected at each

site for each target species if statistical comparisons with the target analyte SVs

are required in the state monitoring programs. The statistical advantages of

replicate sampling are discussed in detail in Section 6.1.2.7. 

6.1.2 Intensive Studies (Tier 2)

The primary aim of intensive studies is to characterize the magnitude and

geographic extent of contamination in harvestable fish and shellfish species at

those screening sites where concentrations of target analytes in tissues were

found to be above selected SVs. Intensive studies should be designed to verify

results of the screening study, to identify specific fish and shellfish species and

size classes for which advisories should be issued, and to determine the geographic extent of the fish contamination. In addition, intensive studies should be

designed to provide data for states to tailor their advisories based on the

consumption habits or sensitivities of specific local fish-consuming subpopulations.

State staff should plan the specific aspects of field collection activities for each

intensive study site after a thorough review of the aims of intensive studies

(Section 2.2) and the fish contaminant data obtained in the screening study. All

the factors that influence sample collection activities should be considered and

specific aspects of each should be documented clearly by the program manager

on the sample request form for each site.

6.1.2.1 Site Selection&

Intensive studies should be conducted at all screening sites where the selected

SV for one or more target analytes was exceeded. The field collection staff

should review a 7.5-minute (1:24,000 scale) USGS hydrologic map of the study

site and all relevant water, sediment, and tissue contaminant data. The site

selection factors evaluated in the screening study (Section 6.1.1.1) must be

reevaluated before initiating intensive study sampling.
6. FIELD PROCEDURES

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States should conduct Tier 2 intensive studies in two phases if program resources

allow. Phase I intensive studies should be more extensive investigations of the

magnitude of tissue contamination at suspect screening sites. Phase II intensive

studies should define the geographic extent of the contamination around these

suspect screening sites in a variety of size (age) classes for each target species.

The field collection staff must evaluate the accessibility of these additional sites

and develop a sampling strategy that is scientifically sound and practicable.

Selection of Phase II sites may be quite straightforward where the source of

pollutant introduction is highly localized or if site-specific hydrologic features

create a significant pollutant sink where chemically contaminated sediments

accumulate and the bioaccumulation potential might be enhanced (U.S. EPA,

1986d). For example, upstream and downstream water quality and sediment

monitoring to bracket point source discharges, outfalls, and regulated disposal

sites showing contaminants from surface runoff or leachate can often be used to

characterize the geographic extent of the contaminated area. Within coves or

small embayments where streams enter large lakes or estuaries, the geographic

extent of contamination may also be characterized via multilocational sampling to

bracket the areas of concern. Such sampling designs are clearly most effective

where the target species are sedentary or of limited mobility (Gilbert, 1987). In

addition, the existence of barriers to migration, such as dams, should be taken

into consideration.

Site selection considerations should also include the number of samples necessary to characterize different waterbody types (lakes, rivers, estuaries, and

coastal marine waters) based on both the hydrodynamics of the waterbody type

including waterbody size as well as the inherent migratory nature of the species

under consideration. Typically, as the size of a waterbody increases (from small

lakes to larger lakes to Great Lakes or from streams, to rivers, to estuaries, to

coastal marine waters), the number of samples that need to be collected to

maintain a selected statistical power (i.e., 70 percent) as well as the number of

sampling stations needed to define the area that should be under advisory both

increase. For example, fish inhabiting relatively small lakes are likely to be

exposed to a relatively homogeneous aquatic environment of contaminant

concentrations. In a riverine, estuarine, or coastal situation, however, the

hydrodynamics of the ecosystem can greatly affect the magnitude and nature of

contamination in the water that fish encounter as they move up and downstream

of areas with distinct nonpoint and point source inputs of contamination. Thus,

the amount of time that any fish spends exposed to the contamination may be

highly variable as compared to the relatively homogeneous exposures that might

occur in smaller, less hydrologically dynamic lake ecosystems.

Overlayed on the hydrodynamic differences of each type of ecosystem and the

spatial distribution of both nonpoint and point sources of pollution that can be

encountered in larger ecosystems are the inherent behavioral differences in fish

and shellfish species with respect to the size of their home range as well as to

whether, at some time or times in their life cycle, they migrate widely to other
6. FIELD PROCEDURES

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more or less contaminated areas. Consider the bluegill sunfish, a common

inhabitant of small lakes and creeks. The home range for this species is typically

less than 0.25 acres (~1,000 m2

) in lakes and does not exceed 28 m in streams

(Carlander, 1969; Hardy, 1978). Smallmouth bass, a riverine species, have a

home range of 500 to 4,500 m2

, but typically migrate up to 45 km (28 miles) (Reid

and Rabeni, 1989; Todd and Rabeni, 1989). In contrast, many Great Lake fish

species, as well as riverine, estuarine, and marine species migrate considerable

distances during spawning periods. Several Great Lakes species also move

upstream considerable distances into tributary rivers to spawn. Lake trout in the

Great Lakes have been found to migrate up to 300 km (186 miles) with larger fish

migrating 300 miles (483 km) (Daly et al., 1962; Mills, 1971; Willers, 1991). For

many marine species, estuaries are the spawning areas for the adults and nursery

areas for the developing juveniles, who eventually travel offshore as adults and

return again to the estuaries to spawn. For these species, migratory or seasonal

movements both from inshore to offshore areas and north and south migrations

along the coasts can take place. Obviously, the number of samples needed to

define an area under advisory for bluegill sunfish inhabiting a relatively

homogeneous environment with respect to contaminant concentrations is quite

different from that required for the more mobile species like the smallmouth bass

and lake trout.

For shellfish, similar considerations are necessary. Bivalve molluscs like the

oyster or mussel cement themselves to hard substrate as young spat and are

unable to move away from pollution effects once they have settled out of the

water column. Although clams and scallop species are slightly more mobile, they

also typically stay in the general area in which they first settled out of the water

column. For crustaceans like the blue crab and lobsters, however, movements

both into and out of estuaries as well as into deeper water offshore are possible.

As the complexity of the hydrodynamics of an ecosystem increases and the

mobility of the target species increases, so too does the number of samples and

the number of sampling stations required to delineate the area where

contaminated individuals may be encountered by the fishing public. 

6.1.2.2 Target Species and Size Class Selection&

Whenever possible, the target species found in the screening study to have

elevated tissue concentrations of one or more of the target analytes should be

resampled in the intensive study. Recommended target species for freshwater

sites are listed in Tables 3-1, 3-2, and 3-4; target species for estuarine/marine

waters are listed in Tables 3-10 through 3-12 for Atlantic Coast estuaries, in Table

3-13 for Gulf Coast estuaries, and in Tables 3-14 through 3-16 for Pacific Coast

estuaries. If the target species used in the screening study are not collected in

sufficient numbers, alternative target species should be selected using criteria

provided in Section 3.2. The alternative target species should be specified on the

sample request form.
6. FIELD PROCEDURES

6-25

For Phase I intensive studies, states should collect replicate composite samples

of one size class for each target species and focus sampling on larger individuals

commonly harvested by the local population (as appropriate). If contamination of

this target size class is high, Phase II studies should include collection of replicate

composite samples of three size classes within each target species.

EPA recognizes that resource limitations may influence the sampling strategy

selected by a state. If monitoring resources are limited for intensive studies,

states may determine that it is more resource-efficient to collect replicate

composite samples of three size classes (as recommended for Phase II studies)

during Phase I sampling rather than revisit the site at a later time to conduct

Phase II intensive studies. In this way, the state may save resources by reducing

field sampling costs associated with Phase II intensive studies.

By sampling three size (age) classes, states collect data on the target species that

may provide them with additional risk management options. If contaminant

concentrations are positively correlated with fish and shellfish size, frequent

consumption of smaller (less contaminated) individuals may be acceptable even

though consumption of larger individuals may be restricted by a consumption

advisory. In this way, states can tailor an advisory to protect human health and

still allow restricted use of the fishery resource. Many Great Lakes states have

used size (age) class data to allow smaller individuals within a given target

species to remain fishable while larger individuals are placed under an advisory.

6.1.2.3 Target Analyte Selection&

Ideally, Phase I intensive studies should include only those target analytes found

in the screening study to be present in fish and shellfish tissue at concentrations

exceeding selected SVs (Section 5.2). Phase II studies should include only those

target analytes found in Phase I intensive studies to be present at concentrations

exceeding SVs. In most cases, the number of target analytes evaluated in

Phase I and II intensive studies will be significantly smaller than the number

evaluated in screening studies.

6.1.2.4 Target Analyte Screening Values&

Target analyte SVs used in screening studies should also be used in Phase I and

II intensive studies. Specific methods used to calculate SVs for noncarcinogenic

and carcinogenic target analytes, including examples of SVs calculated for various

exposure scenarios, are given in Section 5.1.

6.1.2.5 Sampling Times&

To the extent that program resources allow, sampling in intensive studies should

be conducted during the same period or periods during which screening studies

were conducted (i.e., when the target species are most frequently harvested for

consumption) and should be conducted preferably within 1 year of the screening
6. FIELD PROCEDURES

6-26

studies. In some cases, it may be best to combine Phase I and Phase II sampling

to decrease both the time required to obtain adequate data for issuance of

specific advice relative to species, size classes, and geographic extent and/or the

monitoring costs entailed in revisiting the site (see Section 6.1.2.2).

States should follow the general guidance provided in Section 6.1.1.5 for

recommended sampling times. The actual sampling period and rationale for its

selection should be documented fully for Phase I and II studies.

6.1.2.6 Sample Type&

Composite samples of fish fillets or the edible portions of shellfish are recommended for analysis of target analytes in intensive studies. The general guidance

in Section 6.1.1.6 should be followed to prepare composite samples for each

target species. In addition, separate composite samples may be prepared for

selected size (age) classes within each target species, particularly in Phase II

studies after tissue contamination has been verified in Phase I studies. Because

the number of replicate composite samples and the number of fish and shellfish

per composite required to test whether the site-specific mean contaminant

concentration exceeds the selected SV are intimately related, both will be

discussed in the next section. 

Note: The same number of individual organisms should be used to prepare all

replicate composite samples for a given target species at a given site. If this

number is outside the recommended range, documentation should be provided.

Recommended sample preparation procedures are discussed in Section 7.2.

States interested in analyzing target analyte residues in individual fish or shellfish

samples should review information presented in Appendix C.

6.1.2.7 Replicate Samples&

In intensive studies (Phases I and II), EPA recommends that states analyze

replicate composite samples of each target species at each sampling site.

Replicate composite samples should be as similar to each other as possible. In

addition to being members of the same species, individuals within each composite

should be of similar length (size) (see Section 6.1.1.6). The relative difference

between the average length (size) of individuals within any composite sample

from a given site and the average of the average lengths (sizes) of individuals in

all composite samples from that site should not exceed 10 percent (U.S. EPA,

1990d). To determine this, states should first calculate the average length of the

target species fish constituting each composite replicate sample from a site.

Then, states should take the average of these averages for the site. In the

following example, the average of the average lengths of individuals (±10 percent)

in five replicate composite samples is calculated to be 310 (±31) mm.
6. FIELD PROCEDURES

6-27

Replicate

Average Length of Individual

Fish in Composite Sample (mm)

1

2

3

4

5

 300

 320

 330

 280

 320

Average of the average length (±10%) = 310 (±31) mm.

Therefore, the acceptable range for the average length of individual composite

samples is 279 to 341 mm, and the average length of individual fish in each of the

five replicate composites shown above falls within the acceptable average size

range.

All replicate composite samples for a given sampling site should be collected

within no more than 1 week of each other so that temporal changes in target

analyte concentrations associated with the reproductive cycle of the target

species are minimized.

6.1.2.7.1 Guidelines for Determining Sample Sizes&This section provides

general guidelines for estimating the number of replicate composite samples per

site (n) and the number of individuals per composite (m) required to test the null

hypothesis that the mean target analyte concentration of replicate composite

samples at a site is equal to the SV versus the alternative hypothesis that the

mean target analyte concentration is greater than the SV. These guidelines are

applicable to any target species and any target analyte.

Note: It is not possible to recommend a single set of sample size requirements

(e.g., number of replicate composite samples per site and the number of

individuals per composite sample) for all fish and shellfish contaminant monitoring

studies. Rather, EPA presents a more general approach to sample size

determination that is both scientifically defensible and cost-effective. At each site,

states must determine the appropriate number of replicate composite samples

and of individuals per composite sample based on 

& Site-specific estimations of the population variance of the target analyte

concentration

& Fisheries management considerations

& Statistical power consideration.

If the population variance of the target analyte concentrations at a site is small,

fewer replicate composite samples and/or fewer individuals per composite sample

may be required to test the null hypothesis of interest with the desired statistical
6. FIELD PROCEDURES

6-28

power. In this case, using sample sizes that are larger than required to achieve

the desired statistical power would not be cost-effective.

Alternatively, suppose EPA recommended sample sizes based on an analyte

concentration with a population variance that is smaller than that of the target

analyte. In this case, the EPA-recommended sample size requirements may be

inadequate to test the null hypothesis of interest at the statistical power level

selected by the state. Therefore, EPA recommends an approach that provides

the flexibility to sample less in those waters where the target analyte concentrations are less variable, thereby reserving sampling resources for those sitespecific situations where the population variance of the target analyte tissue

concentration is greater.

EPA recommends the following statistical model, which assumes that zi

 is the

contaminant concentration of the ith replicate composite sample at the site of

interest where i=1,2,3,...,n and, furthermore, that each replicate composite sample

is comprised of m individual fish fillets of equal mass. Let z be the mean target  ̄

analyte concentration of observed replicate composite samples at a site. Ignoring

measurement error, the variance of z is  ̄

Var(z) =  ̄ )2

/(nm) (6-1)

where

)2 = Population variance

n = Number of replicate composite samples

m = Number of individual samples in each composite sample.

To test the null hypothesis that the mean target analyte concentration across the

n replicate composite samples is equal to the SV versus the alternative hypothesis

that the mean target analyte concentration is greater than the SV, the estimate of

the Var(z), s  ̄ 2

, is

s2

 = [((zi  z) ̄ 2

] / [n(n  1)] (6-2)

where the summation occurs over the n composite samples. Under the null

hypothesis, the following statistic

(z  ̄  SV) / s (6-3)

has a Student-t distribution with (n  1) degrees of freedom (Cochran, 1977; Kish,

1965). The degrees of freedom are one less than the number of composite

samples.

Note: Use of a single composite sample precludes estimating the variability of

the mean target analyte concentration. The estimator s2

 can only be calculated

with at least two (but preferably three or more) replicate composite samples.
6. FIELD PROCEDURES

6-29

An optimal sampling design would specify the minimum number of replicate

composite samples (n) and of individuals per composite (m) required to detect a

minimum difference between the selected SV and the mean target analyte

concentration of replicate composite samples at a site. Design characteristics

necessary to estimate the optimal sampling design include

 Minimum detectable difference between the site-specific mean target analyte

concentration and the selected SV

 Power of the hypothesis test (i.e., the probability of detecting a true difference

when one exists)

 Level of significance (i.e., the probability of rejecting the null hypothesis of no

difference between the site-specific mean target analyte concentration and the

SV when a difference does not exist)

 Population variance, )2

 (i.e., the variance in target analyte concentrations

among individuals from the same species, which the statistician often must

estimate from prior information)

 Cost components (including fixed costs and variable sample collection,

preparation, and analysis costs).

In the absence of such design specifications, guidance for selecting the number

of replicate composite samples at each site and the number of fish per composite

sample is provided. This guidance is based on an investigation of the precision

of the estimate of )2

/nm and of statistical power.

Note: Under optimal field and laboratory conditions, at least two replicate

composite samples are required at each site for variance estimation. To minimize

the risk of a destroyed or contaminated composite sample precluding the sitespecific statistical analysis, a minimum of three replicate composite samples

should be collected at each site if possible. Because three replicate composite

samples provide only two degrees of freedom for hypothesis testing, additional

replicate composite samples are recommended.

The stability of the estimated standard error of z must also be considered because  ̄

this estimated standard error is the denominator of the statistic for testing the null

hypothesis of interest. A measure of the stability of an estimate is its statistical

precision. The assumption is made that the zi

's come from a normal distribution,

and then the standard error of is defined as a product of and a function σ 2 /nm σ 2

of n (the number of replicate composite samples) and m (the number of fish per

composite). A fortunate aspect of composite sampling is that the composite target

analyte concentrations tend to be normally distributed via the Central Limit

Theorem. This formulation is used to determine which combinations of n and m

are associated with a more precise estimate of )2

/nm.
6. FIELD PROCEDURES

6-30

Table 6-1. Values of for Various Combinations of n and m 2

n m (n 1). 2 2

1/ 2

−





 





No. of replicate

composite

samples (n)

Number of fish per composite sample (m)

3 4 5 6 7 8 9 10 12 15

3

4

5

6

7

10

15

0.111

0.068

0.047

0.035

0.027

0.016

0.008

0.083

0.051

0.035

0.026

0.021

0.012

0.006

0.067

0.041

0.028

0.021

0.016

0.009

0.005

0.056

0.034

0.024

0.018

0.014

0.008

0.004

0.048

0.029

0.020

0.015

0.012

0.007

0.004

0.042

0.026

0.018

0.013

0.010

0.006

0.003

0.037

0.023

0.016

0.012

0.009

0.005

0.003

0.033

0.020

0.014

0.011

0.008

0.005

0.003

0.028

0.017

0.012

0.009

0.007

0.004

0.002

0.022

0.014

0.009

0.007

0.005

0.003

0.002

Modifying Cochran (1963) to reflect the normality assumption and the sampling

design of n replicate composite samples and m fish per composite sample, the

function of n and m of interest is shown in square brackets:

se (6-4)

nm

2

n m (n 1)

2

2 2

1/2

σ σ 



 



 = −





 





Table 6-1 provides values of this function for various combinations of m and n.

The data presented in Table 6-1 suggest that, as either n or m increases, the

standard error of decreases. The advantage of increasing the number of σ 2 /nm

replicate composite samples can be described in terms of this standard error. For

example, the standard error of from a sample design of five replicate σ 2 /nm

composite samples and six fish per composite (0.024) will be more than 50

percent smaller than that from a sample design of three replicate composite

samples and six fish per composite (0.056). In general, holding the number of fish

per composite fixed, the standard error of estimated from five replicate σ 2 /nm

samples will be about 50 percent smaller than that estimated from three replicate

samples.

The data in Table 6-1 also suggest that greater precision in the estimated

standard error of z is gained by increasing the number of replicate samples (n)  ̄

than by increasing the number of fish per composite (m). If the total number of

individual fish caught at a site, for example, is fixed at 50 fish, then, with a design

of 10 replicate samples of 5 fish each, the value of the function of n and m in

Table 6-1 is 0.009; with 5 replicate samples of 10 fish each, the value is 0.014.

Thus, there is greater precision in the estimated standard error of z associated  ̄

with the first design as compared with the second design.

Two assumptions are made to examine the statistical power of the test of the null

hypothesis of interest. First, it is assumed that the true mean of the site-specific

composite target analyte concentrations (μ) is either 10 percent, 25 percent, or 50

percent higher than the screening value. Second, it is presumed that a factor

similar to a coefficient of variation, the ratio of the estimated population standard

deviation to the screening value (i.e., )/SV), is 50, 75 or 100 percent. Nine
6. FIELD PROCEDURES

6-31

scenarios result from joint consideration of these two assumptions. The power of

the test of the null hypothesis that the mean composite target analyte

concentration at a site is equal to the SV versus the alternative hypothesis that the

mean target analyte concentration is greater than the SV is estimated under each

set of assumptions. Estimates of the statistical power for six of the nine scenarios

are shown in Table 6-2.

Power estimates for the three scenarios where the true mean of the site-specific

composite target analyte concentration was assumed to be only 10 percent higher

than the screening value are not presented. The power to detect this small

difference was very poor: for 242 of the resulting 270 combinations of n and m,

the power was less than 50 percent.

Several observations can be made concerning the data in Table 6-2. Note: The

statistical power increases as either n (number of replicate composite samples)

or m (number of fish per composite) increases. However, greater power is

achieved by increasing the number of replicate composite samples as opposed

to increasing the number of fish per composite. Furthermore, if the number of

replicate composite samples per site and the number of fish per composite are

held constant, then, as the ratio of the estimated population standard deviation to

the SV increases (i.e., )/SV), the statistical power decreases. Higher variability

in the true population of target analyte concentration in fish will require more

samples to detect a difference between the mean target analyte concentration

and the SV.

States may use these tables as a starting point for setting the number of replicate

composite samples per site and the number of fish per composite in their fish and

shellfish contaminant monitoring studies. The assumption regarding the ratio of

the estimated population standard deviation to the SV presented in Sections A

and D of Table 6-2 is unrealistic for some fish and shellfish populations. Data in

Sections C through F, which reflect more realistic assumptions concerning the

estimated population standard deviation, show that states will be able to detect

only large differences between the site-specific mean target analyte concentrations and the selected SV. Specifically, if the assumed ratio of the estimated

population standard deviation to the SV is 1.0, using five replicate composite

samples and six to seven fish per composite sample, the power to detect a 50

percent increase over the SV is between 70 and 80 percent. However, when the

number of fish per composite increases to 8 to 10, the power increases by about

10 percentage points. In comparison, the power to detect a 25 percent increase

over the SV is less than 50 percent.

Table 6-2 shows that a statistical power level of (at least) 70 percent is attainable

for moderate values of m and n, as long as the ratio )/SV is not large and/or the

desired detectable difference between the target analyte concentration and the

SV is not too small. 
6. FIELD PROCEDURES

6-32

Table 6-2. Estimates of Statistical Power of Hypothesis of Interest Under

Specified Assumptions

No. of Replicate

Composite

Samples (n)

Number of Fish Per Composite (m)

3 4 5 6 7 8 9 10 12 15

A. Ratio of )/SV = 0.5 and μ = 1.5 x SV:

3

4

5

6

7

8

9

10

15

5

8

9

9

9

9

9

9

9

6

9

9

9

9

9

9

9

9

7

9

9

9

9

9

9

9

9

8

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

9

B. Ratio of )/SV = 0.75 and μ = 1.5 x SV:

3

4

5

6

7

8

9

10

15

%

%

6

7

8

8

9

9

9

%

6

7

8

9

9

9

9

9

%

7

8

9

9

9

9

9

9

%

7

8

9

9

9

9

9

9

5

8

9

9

9

9

9

9

9

6

8

9

9

9

9

9

9

9

6

9

9

9

9

9

9

9

9

7

9

9

9

9

9

9

9

9

7

9

9

9

9

9

9

9

9

8

9

9

9

9

9

9

9

9

C. Ratio of )/SV = 1.0 and μ = 1.5 x SV:

3

4

5

6

7

8

9

10

15

%

%

%

5

6

7

7

8

9

%

%

5

6

7

8

8

8

9

%

%

6

7

8

8

9

9

9

%

5

7

8

8

9

9

9

9

%

6

7

8

9

9

9

9

9

%

6

8

8

9

9

9

9

9

%

7

8

9

9

9

9

9

9

%

7

8

9

9

9

9

9

9

5

8

9

9

9

9

9

9

9

6

8

9

9

9

9

9

9

9

D. Ratio of )/SV = 0.5 and μ = 1.25 x SV:

3

4

5

6

7

8

9

10

15

%

%

%

5

6

7

7

8

9

%

%

5

6

7

8

8

8

9

%

%

6

7

8

8

9

9

9

%

5

7

8

8

9

9

9

9

%

6

7

8

9

9

9

9

9

%

6

8

8

9

9

9

9

9

%

7

8

9

9

9

9

9

9

%

7

8

9

9

9

9

9

9

5

8

9

9

9

9

9

9

9

6

8

9

9

9

9

9

9

9

E. Ratio of )/SV =0.75 and μ = 1.25 x SV:

3

4

5

6

7

8

9

10

15

%

%

%

%

%

%

%

%

6

%

%

%

%

%

%

5

6

7

%

%

%

%

5

5

6

6

8

%

%

%

%

5

6

7

7

9

%

%

%

5

6

7

7

8

9

%

%

%

6

6

7

8

8

9

%

%

5

6

7

8

8

8

9

%

%

5

7

7

8

8

9

9

%

%

6

7

8

8

9

9

9

%

6

7

8

9

9

9

9

9
6. FIELD PROCEDURES

Table 6-2. (continued)

No. of Replicate

Composite

Samples (n)

Number of Fish Per Composite (m)

3 4 5 6 7 8 9 10 12 15

6-33

F. Ratio of )/SV = 1.0 and μ = 1.25 x SV:

3

4

5

6

7

8

9

10

15

%

%

%

%

%

%

%

%

%

%

%

%

%

%

%

%

%

5

%

%

%

%

%

%

%

%

6

%

%

%

%

%

%

%

5

5

%

%

%

%

%

%

5

5

7

%

%

%

%

%

5

5

6

8

%

%

%

%

5

5

6

6

8

%

%

%

%

5

6

6

7

8

%

%

%

5

6

7

7

8

9

%

%

5

6

7

7

8

8

9

%: Power less than 50 percent.

5: Power between 50 and 60 percent.

6: Power between 60 and 70 percent.

7: Power between 70 and 80 percent.

8: Power between 80 and 90 percent

9: Power greater than 90 percent

One final note on determining the number of replicate composite samples per site

and the number of fish per composite should be emphasized. According to

Section 6.1.2.3, Phase I intensive studies will focus on those target analytes that

exceeded the selected SV used in the screening study. Thus, multiple target

analytes may be under investigation during Phase I intensive studies, and the

population variances of these analytes are likely to differ. Note: States should

use the target analyte that exhibits the largest population variance when selecting

the number of replicate composite samples per site and the number of fish per

composite. This conservative approach supports use of the data in Section B of

Table 6-2 where the ratio of )/SV is twice that of the data in Section A. States

may estimate population variances from historic fish contaminant data or from

composite data as described by U.S. EPA (1989d). This estimate of )2

 can be

used to determine whether the sampling design (i.e., number of replicate

composite samples [n] and number of individuals per composite [m]) should be

modified to achieve a desired statistical power.

Table 6-3 summarizes some observed ratios ()/SV) of selected target analytes.

These values were estimated from composite samples of siscowet trout and lake

trout collected and analyzed by the Great Lakes Indian Fish and Wildlife

Commission in a study funded by the Administration for Native Americans.

Table 6-3. Observed Ratios ()/SV) of Selected Target Analytes

Target Species

Total PCB

SV=0.02 ppm

Observed )/SV (Mean)

Toxaphene

SV=0.0363 ppm

Heptachlor Epoxide

SV=0.00439 ppm

Siscowet trout 4.08 (1.01) 7.07 (2.18) 0.68 (0.01)

Lake trout 10.70 (0.47) 3.01 (0.38) 0.93 (0.007)

Source: Personal communication, Kory Groetsch, Great Lakes Indian Fish and Wildlife

Commission, Odana, WI, with Elvessa Aragon, Research Triangle Institute, Research

Triangle Park, NC, May 10, 2000.

SV = EPA default value for recreational fishers.
6. FIELD PROCEDURES

6-34

Consider a study of heptachlor epoxide concentrations in lake trout. The

observed ratio ()/SV) is close to 1.0 and the observed mean is approximately

1.5 x SV. To determine the appropriate values of n and m, we look at Section C

of Table 6-2. To achieve statistical power between 80 and 90 percent, the

combination of n and m that requires the smallest number of individual fish is

n=10 and m=3. Ten replicate composite samples, each with three fish, will

provide between 80 and 90 percent power for detecting a mean heptachlor

epoxide concentration that is higher than the SV, if the difference truly exists.

Other combinations of n and m might be more desirable. For instance, if the cost

of analyzing composite samples is much higher than the cost of compositing

individual fish, a combination that yields fewer replicate composite samples (say,

n=5 and m=8, or n=6 and m=6) may be chosen. For siscowet trout, the observed

ratio ()/SV) is close to 0.75 while the observed mean is approximately 2.25 x SV.

A comparison of the combinations of n and m in Sections B and E (for

)/SV = 0.75) shows that higher values of n and m are required to detect a

difference at the same level of statistical power. For instance, in Section B, where

μ = 1.5 x SV, the smallest number of individual fish needed to achieve 80 to

90 percent power is given by n=7 and m=3. In Section E, where μ=1.25 x SV, the

combination of n=15 and m=5 achieves 80 to 90 percent power. For the same

level of power and the same )/SV, detecting a larger difference between the SV

and the true mean concentration requires larger sample sizes (n or m or both).

After states have implemented their fish and shellfish contaminant monitoring

program, collected data on cost and variance components, and addressed other

design considerations, they may want to consider using an optimal composite

sampling protocol as described in Rohlf et al. (1991) for refining their sampling

design. An optimal sampling design is desirable because it detects a specified

minimum difference between the site-specific mean contaminant concentration

and the SV at minimum cost.

6.1.2.7.2 Comparison of Target Analyte Concentrations with Screening

Values for Issuing Fish Advisories&Using the statistical model described in

Section 6.1.2.7.1, target analyte concentrations from replicate composite samples

at a particular site can be compared to screening values using a t-test. Assume

that zi

 is the contaminant concentration of the ith replicate composite sample at

the site of interest where i=1,2,3,...,n and, furthermore, that each replicate

composite sample comprises m individual fish fillets of equal mass. To test the

null hypothesis that the mean target analyte concentration across the n replicate

composite samples is equal to the SV versus the alternative hypothesis that the

mean target analyte concentration is greater than the SV, perform the following

steps:

1. Calculate z, the mean target analyte concentration of observed replicate  ̄

composite samples at a site: 

z =  ̄ (zi

 / n

where the summation occurs over the n composite samples.
6. FIELD PROCEDURES

6-35

2. Calculate the estimate of the Var(z), s  ̄ 2

 :

s2

 = [((zi z) ̄ 2] / [n(n  1)] 

where the summation occurs over the n composite samples. 

3. Calculate the test statistic:

tc = (z  ̄  SV) / s

4. The null hypothesis of no difference is rejected in favor of the alternative

hypothesis of exceedance if 

 tc > t,n-1

where t,n-1 is the tabulated value of the Student-t distribution corresponding

to level of significance  and n-1 degrees of freedom. Note that the inequality

is in one direction (>) since it is exceedance of the SV that is of interest.

When several sites are sampled and/or fish of different size ranges are collected,

it is important to conduct the test separately at each site and for each size range.

Combining sites or size ranges introduces variance components that are not

accounted for in this procedure. The variance estimate may be larger with the

additional sources of variability, and more replicate samples may be needed to

detect a significant overall exceedance of the SV.

Example

Samples of siscowet trout were collected by the Great Lakes Indian Fish and

Wildlife Commission and composited according to the guidelines discussed in this

document. Composites of 12 fish were prepared, and four replicate samples of

each of four size classes were analyzed for total mercury, PCBs, and a suite of

chlorinated pesticides. Following is a summary of the test for exceedance of the

SV for hexachlorobenzene (SV=0.025 ppm) based on the recreational fish

consumption default value. 

At the 5 percent level of significance the critical value of the Student-t distribution

with three degrees of freedom is 2.353. All of the test statistic values are less than

the critical value. The mean levels of hexachlorobenzene in the four size ranges

of siscowet trout are less than the SV, so no fish advisory is needed.
6. FIELD PROCEDURES

6-36

Size Range

(in.)

No. of

Replicate

Samples

(n)

No. of

Fish per

Composite

(m)

Composite

Measurements

of HCB 

(ppm)

Mean

(Estimated

Standard

Deviation)

Test

Statistic

17.0-18.0 4 12 0.00419

0.00507

0.00483

0.00405

4.53x10-3

(2.46x10-4)

-83.21

19.5-20.5 4 12 0.00604

0.00780

0.00925

0.00990

8.25x10-3

(8.57x10-4)

-19.54

22.0-23.0 4 12 0.01800

0.01808

0.01868

0.02389

1.97x10-2)

(1.42x10-3)

-3.73

24.5-25.5 4 12 0.01050

0.00960

0.00850

0.01090

9.88x10-3

(5.33x10-4)

-28.37

HCB=Hexachlorobenzene.

6.1.2.7.3 Comparison of Target Analyte Concentrations with Screening

Values for Rescinding Fish Advisories&The comparison of mean target analyte

concentrations to the screening values must be statistically based when

considering rescinding a fish advisory. Statistical tests are constructed to control

the Type I and Type II errors. The Type I error is defined as rejecting the null

hypothesis (based on the evidence from the data) even though it is really true.

The Type II error is defined as failing to reject the null hypothesis even though it

is really false. In the context of the null and alternative hypotheses presented in

the previous section, the Type I error is concluding that the mean target analyte

concentration exceeds the SV when in fact it does not. The state concludes that

there is a need to issue a fish advisory and proceeds to issue one, albeit

unnecessarily. The Type II error is concluding that the mean target analyte

concentration tissue residue level does not exceed the SV when in fact it does.

The state decides that the mean target analyte concentration is no longer

endangering the public health, so the fish advisory is rescinded. The implications

of such errors may be costly; a Type II error in this case will put the public at risk

without their knowledge. The Type I error is controlled by setting the level of

significance to a small value, and the Type II error is controlled by increasing the

power of the test. Both error types can be controlled simultaneously by increasing

the sample sizes (n or m or both). 

There are two basic statistical questions that must be answered before a fish

advisory is rescinded: 

 Is the screening value still being exceeded?

 If the screening value is no longer being exceeded, can the target analyte

concentrations be expected to remain below the screening value?
6. FIELD PROCEDURES

6-37

The first question may be answered with the t-test described in the previous

section. The second question may be answered by monitoring the target analyte

concentrations long enough to observe a downward trend or a constant trend

below the screening value. The simple approach would be to obtain replicate

composite samples each year and test for exceedance of the screening value.

(Section 6.1.1.5 recommends that screening be done biennially or at least once

every 5 years. "Year" then signifies the years when screening is performed.) If

the screening value is no longer being exceeded in year X, the state should

continue obtaining replicate samples for at least one more year. The state should

then test the differences between the tissue residue levels at years X-1, X, and

X+1. Significant differences between the levels, especially between years X-1 and

X, as well as between years X-1 and X+1, allows verification that the decrease in

the target analyte concentration below the screening value at year X was not by

chance. Appendix N discusses some statistical methods for comparing samples

at different time points. 

It is recommended that the yearly studies be as similar in study design as

possible. Introducing changes in the study design will add more sources of

variability and may necessitate increasing the number of replicate samples or

accounting for the additional variance components in the statistical methods used.

6.1.2.7.4 Issuing Statewide Advisories&In addition to issuing fish consumption

advisories for individual waterbodies, 18 states have also issued blanket statewide

advisories for certain types of waterbodies within their jurisdictions (U.S. EPA,

1999c). States have issued statewide advisories for their freshwater lakes and/or

rivers and their coastal waters, which can include estuaries and/or coastal marine

waters. States often issue statewide advisories for certain waterbody types to

warn the public of the potential for widespread contamination of certain species

of fish or shellfish in these waterbodies. In these cases, the state has typically

found a level of contamination of a specific pollutant in a particular fish species

over a relatively wide geographic area that warrants advising the public of the

situation. A state often issues a statewide advisory when, for example, it has

many lakes that need to be monitored but has limited resources to collect fish

(can sample only four or five lakes per year). If the state has even 100 lakes that

need monitoring at the level of resources available, it could take 10 to 20 years

to adequately monitor all 100 lakes. As an alternative, some states monitor a

small percentage of their lakes and, based on the level of contamination found,

many have determined that a statewide advisory should be issued to be

conservative with respect to protection of public health. Methylmercury, because

it is dispersed and transported via the atmosphere, is the leading pollutant

responsible for the issuance of statewide advisories in 15 states, although PCBs,

dioxins/furans, cadmium, chlordane, mirex, and DDT are also responsible for

statewide advisories in a smaller number of states. Assuming that the levels of

contamination are determined based on the fish compositing guidelines in this

document, the biggest question is determining which waterbodies to monitor.

Finding a "representative" sample of waterbodies is a daunting task since there

are many different ways to determine representativeness: size of waterbody,
6. FIELD PROCEDURES

6-38

species of interest, dynamics of dispersion of pollutants of interest, or

geographical location. Taking a simple random sample of lakes may not achieve

sufficient coverage, whereas taking a stratified random sample approach may

require more lakes be sampled than can be afforded. A conservative approach

may be to look at the "worst case scenario". States may decide to sample the

lakes that are believed to have the highest levels of pollutants, based on historical

contaminant data, current water and sediment sampling results, or other

variables. Another approach would be to select one or two of the factors

described above ("representativeness"), stratify the lakes according to these

factors, and select a random sample within each stratum. The set of factors for

stratification may change every few years or so if it is deemed that some other

factors are becoming more indicative of the levels of contamination.

6.2 SAMPLE COLLECTION

Sample collection activities should be initiated in the field only after an approved

sampling plan has been developed. This section discusses recommended

sampling equipment and its use, considerations for ensuring preservation of

sample integrity, and field recordkeeping and chain-of-custody procedures

associated with sample processing, preservation, and shipping.

6.2.1 Sampling Equipment and Use

In response to the variations in environmental conditions and target species of

interest, fisheries biologists have had to devise sampling methods that are

intrinsically selective for certain species and sizes of fish and shellfish (Versar,

1982). Although this selectivity can be a hindrance in an investigation of

community structure, it is not a problem where tissue contaminant analysis is of

concern because tissue contaminant data can best be compared only if factors

such as differences in taxa and size are minimized.

Collection methods can be divided into two major categories, active and passive.

Each collection method has advantages and disadvantages. Various types of

sampling equipment, their use, and their advantages and disadvantages are

summarized in Table 6-4 for fish and in Table 6-5 for shellfish. Note: Either

active or passive collection methods may be used as long as the methods

selected result in collection of a representative fish sample of the type consumed

by local sport and subsistence fishers.

A basic checklist of field sampling equipment and supplies is shown in Table 6-6.

Safety considerations associated with the use of a boat in sample collection

activities are summarized in Table 6-7.

6.2.1.1 Active Collection&

Active collection methods employ a wide variety of sampling techniques and

devices. Devices for fish sampling include electroshocking units, seines, trawls,
6. FIELD PROCEDURES

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Table 6-4. Summary of Fish Sampling Equipment

Device Use Advantages Disadvantages

ACTIVE METHODS

Electrofishing Shallow rivers, lakes, and streams. Most efficiency nonselection method. Minimal

damage to fish. Adaptable to a number ofsampling conditions (e.g., boat, wading,shorelines). Particularly useful at sites where otheractive methods cannot be used (e.g., around snagsand irregular bottom contours).

Nonselective%stuns or kills most fish. Cannot beused in brackish, salt, or extremely soft water. Requires extensive operator training. DANGEROUS when not used properly.

Seines Shallow rivers, lakes, and streams. Shoreline

areas of estuaries.

Relatively inexpensive and easily operated. Meshsize selection available for target species.

Cannot be used in deep water or over substrateswith an irregular contour. Not completely efficientas fish can evade the net during seining operation.

Trawls Various sizes can be used from boats in moderate

to deep open bodies of water (10 to >70 m depths).

Effective in deep waters not accessible by othermethods. Allows collection of a large number ofsamples.

Requires boat and trained operators.

Angling Generally species selective involving use of hook

and line.

Most selective method. Does not require use oflarge number of personnel or expensiveequipment.

Inefficient and not dependable.

Purchasing specimensfrom commercial fishers

Only in areas where target species arecommercially harvested.

Most cost-effective and efficient means of obtainingcommercially valuable species from harvestedwaters.

Limited use; commercially harvested areas may notinclude sampling sites chosen for fish contaminantmonitoring. The field collection staff shouldaccompany the commercial fishers and shouldremove the required samples from the collectiondevice. This will ensure the proper handling of thespecimens and accurate recording of the collectiontime and sampling location.

PASSIVE METHODS

Gill nets Lakes, rivers, and estuaries. Where fish

movement can be expected or anticipated.

Effective for collecting pelagic fish species. Relatively easy to operate. Requires less fishingeffort than active methods. Selectivity can becontrolled by varying mesh size.

Not effective for bottom-dwelling fish or populationsthat do not exhibit movement patterns. Nets proneto tangling or damage by large and sharp spinedfish. Gill nets will kill captured specimens, which,when left for extended periods, may undergophysiological changes.

Trammel nets Lakes, rivers, and estuaries. Where fish

movement can be expected or anticipated. Frequently used where fish may be scared into thenet.

Slightly more efficient than a straight gill net. (Same as for gill nets.) Tangling problems may be

more severe. Method of scaring fish into netrequires more personnel or possibly boats in deepwater areas.

Hoop, Fyke and PoundNets

Shallow rivers, lakes, and estuaries when currentsare present or when movements of fish arepredictable. Frequently used in commercialoperations.

Unattended operation. Very efficient in regard tolong-term return and expended effort. Particularlyuseful in areas where active methods areimpractical.

Inefficient for short term. Difficult to set up andmaintain.

D-Traps

Used for long-term capture of slow-moving fish,particularly bottom species. Can be used in allenvironments.

Easy to operate and set. Unattended operation. Particularly useful for capturing bottom-dwellingorganisms in deep waters or other types ofinaccessible areas. Relatively inexpensive%oftencan be hand made.

Efficiency is highly variable. Not effective forpelagic fish or fish that are visually oriented. Lessefficient for all species when water is clear ratherthan turbid. Not a good choice for a primarysampling technique, but available as backup forother methods.

Source: Versar, 1982.
6. FIELD PROCEDURES

6-40

Table 6-5. Summary of Shellfish Sampling Equipment

Device Use Advantages Disadvantages

ACTIVE METHODS

Seines Shallow shoreline areas of estuaries. Relatively inexpensive and easily operated. Mesh

size selection available for target crustaceanspecies (e.g., shrimp and crabs).

Cannot be used in deep water or over substrateswith an irregular contour. Not completely efficientas crustaceans can evade the net during seiningoperation.

Trawls Various sizes can be used from boats in moderate

to deep open bodies of water (10 to >70 m depths).

Effective in deeper waters not accessible by othermethods. Allows collection of a large number ofsamples.

Requires boat and trained operators.

Mechanical grabs

Double-pole-operatedgrab buckets

Used from boat or pier. Most useful in shallowwater areas less than 6 m deep including lakes,rivers, and estuaries.

Very efficiency means of sampling bivalves (e.g.,clams and oysters) that are located on or buried inbottom sediments.

At depths greater than 6 m, the pole-operateddevices become difficult to operate manually.

Tongs or doublehandled grab sampler Most useful in shallow water, lakes, rivers, and estuaries. Generally used from a boat. Very efficient means of sampling oysters, clams, and scallops. Collection of surrounding or

overlying sediments is not required and the jawsare generally open baskets. This reduces theweight of the device and allows the washing ofcollected specimens to remove sediments.

At depths greater than 6 m, the pole-operateddevices become difficult to operate manually.

Line or cable-operatedgrab buckets

Ekman grab Used from boat or pier to sample soft to semisoft

substrates.

Can be used in water of varying depths in lakes,rivers, and estuaries.

Possible incomplete closure of jaws can result insample loss. Must be repeatedly retrieved anddeployed. Grab is small and is not particularlyeffective in collecting large bivalves (calms andoysters).

Petersen grab Deep lakes, rivers, and estuaries for sampling most

substrates.

Large sample is obtained; grab can penetrate mostsubstrates.

Grab is heavy, may require which for deployment. Possible incomplete closure of jaws can result issample loss. Must be repeatedly retrieved anddeployed.

Ponar grab Deep lakes, rivers, and estuaries for sampling

sand, silt, or clay substrates.

Most universal grab sampler. Adequate on mostsubstrates. Large sample is obtained intact.

Possible incomplete closure of jaws can result insample loss. Must be repeatedly retrieved anddeployed.

Orange peel grab Deep lakes, rivers, and estuaries for sampling most

substrates.

Designed for sampling hard substrates. Grab is heavy, may require winch for deployment. 

Possible incomplete closure of jaws can result insample loss. Must be repeatedly retrieved anddeployed. Grab is small and not particularlyeffective in collecting large bivalves (clams andoysters).

Biological or hydraulicdredges

Dragged along the bottom of deep waterbodies tocollect large stationary invertebrates.

Qualitative sampling of large area of bottomsubstrate and benthic community. Length of towscan be relatively short if high density of shellfishexists in sampling area.

If the length of the tow is long, it is difficult topinpoint the exact location of the sample collectionarea. Because of the scouring operation of thedredge, bivalve shells may be damaged. Allbivalve specimens should be inspected andindividuals with cracked or damaged shells shouldbe discarded.

(continued)
6. FIELD PROCEDURES

6-41

Table 6-5. (continued)

Device Use Advantages Disadvantages

ACTIVE METHODS (continued)

Scoops, shovels Used in shallow waters accessible by wading or

SCUBA equipment for collection of hard clams(Mercenaria mercenaria) or soft-shell clam (Myaarenaria).

Does not require a boat; sampling can be donefrom shore.

Care must be taken not to damage the shells ofbivalves while digging in substrate.

Scrapers Used in shallow waters accessible by wading or

SCUBA equipment for collection of oysters(Crassostrea virginica) or mussels (Mytilus sp).

Does not require a boat; sampling can be donefrom shore.

Care must be taken not to damage shells ofbivalves while removing them from hard substrate.

Rakes Used in shallow waters accessible by wading or

can be used from a boat.

Does not require a boat; sampling can be doneclose to shore. Can be used in soft sediments tocollect clams or scallops and can also be used todislodge oysters or mussels that are attached tosubmerged objects such as rocks and pier pilings.

Care must be taken not to damage the shells of thebivalves while raking or dislodging them from thesubstrate.

Purchasing specimensfrom commercial fishers

Only in areas where target species arecommercially harvested.

Most cost-effective and efficient means of obtainingbivalves for pollutant analysis from commerciallyharvested waters.

Limited use; commercially harvested areas may notinclude sampling sites chosen for shellfishcontaminant monitoring. The field collection staffshould accompany the commercial fishers andshould remove the required samples from thecollection device. This will ensure the properhandling of the specimens and accurate recordingof the exact collection time and sampling location.

PASSIVE METHODS

D-traps Used for capture of slow-moving crustaceans

(crabs and lobsters) that move about on or justabove the substrate.

Can be used in a variety of environments. Particularly useful for capturing bottom-dwellingorganisms in deep water or other inaccessibleareas. Relatively inexpensive, can be hand made.

Catch efficiency is highly variable. Not a goodchoice for a primary sampling technique, butvaluable as a backup for other methods.
6. FIELD PROCEDURES

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Table 6-6. Checklist of Field Sampling Equipment and Supplies

for Fish and Shellfish Contaminant Monitoring Programs

* Boat supplies

* Fuel supply (primary and auxiliary supply)

* Spare parts repair kit

* Life preservers

* First aid kit (including emergency phone numbers of local hospitals, family contacts

for each member of the sampling team)

* Spare oars

* Nautical charts of sampling site locations

* Collection equipment (e.g., nets, traps, electroshocking device)

* Recordkeeping/documentation supplies

* Field logbook

* Sample request forms

* Specimen identification labels

* Chain-of-Custody (COC) Forms and COC tags or labels

* Indelible pens

* Sample processing equipment and supplies

* Holding trays

* Fish measuring board (metric units)

* Calipers (metric units)

* Shucking knife

* Balance to weigh representative specimens for estimating tissue weight (metric units)

* Aluminum foil (extra heavy duty)

* Freezer tape

* String

* Several sizes of plastic bags for holding individual or composite samples

* Resealable watertight plastic bags for storage of Field Records, COC Forms, and

Sample Request Forms

* Sample preservation and shipping supplies

* Ice (wet ice, blue ice packets, or dry ice)

* Ice chests

* Filament-reinforced tape to seal ice chests for transport to the central processing

laboratory
6. FIELD PROCEDURES

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Table 6-7. Safety Considerations for Field Sampling Using a Boat

 Field collection personnel should not be assigned to duty alone in boats.

 Life preservers should be worn at all times by field collection personnel near the water or

on board boats.

 If electrofishing is the sampling method used, there must be two shutoff switches--one at

the generator and a second on the bow of the boat.

 All deep water sampling should be performed with the aid of an experienced, licensed

boat captain.

 All sampling during nondaylight hours, during severe weather conditions, or during

periods of high water should be avoided or minimized to ensure the safety of field

collection personnel.

 All field collection personnel should be trained in CPR, water safety, boating safety, and

first aid procedures for proper response in the event of an accident. Personnel should

have local emergency numbers readily available for each sampling trip and know the

location of the hospitals or other medical facilities nearest each sampling site.

and angling equipment (hook and line). Rotenone, a chemical piscicide, has been

used extensively to stun fish prior to their collection with seines, trawls, or other

sampling devices. Rotenone has not been found to interfere with the analysis of

the recommended organic target analytes (see Table 4-1) when the

recommended analysis procedures are used. See Section 8 for additional

information on appropriate analysis methods for the recommended organic target

analytes. Devices for shellfish sampling include seines, trawls, mechanical grabs

(e.g., pole- or cable-operated grab buckets and tongs), biological and hydraulic

dredges, scoops and shovels, rakes, and dip nets. Shellfish can also be collected

manually by SCUBA divers. Although active collection requires greater fishing

effort, it is usually more efficient than passive collection for covering a large

number of sites and catching the relatively small number of individuals needed

from each site for tissue analysis (Versar, 1982). Active collection methods are

particularly useful in shallow waters (e.g., streams, lake shorelines, and shallow

coastal areas of estuaries). 

One aspect of sample collection that is of paramount importance is that the

sampling team must ensure the collection of live, intact fish and shellfish for use

in sample analysis for human risk assessment. It is highly desirable to collect

live, intact fish and shellfish that have not been mutilated by the collection gear

and that do not have any skin, shell, or carapace lacerations or fin deterioration

that would allow body fluids to leak out of the specimen or contaminants to pass

into the specimen after collection. For example, some fish collected by electroshocking methods may have ruptured organs due to the electroshocking

procedure. Fish that are found floating dead at a site should not be used for

sample analysis for human risk assessments. For these reasons, EPA recommends that any specimens that show any skin, shell, or carapace lacerations or

fin deterioration of any kind not used for chemical analysis. 
6. FIELD PROCEDURES

6-44

Active collection methods have distinct disadvantages for deep water sampling.

They require more field personnel and more expensive equipment than passive

collection methods. This disadvantage may be offset by coordinating sampling

efforts with commercial fishing efforts. Purchasing fish and shellfish from commercial fishers using active collection devices is acceptable; however, field

sampling staff should accompany the commercial fishers during the collection

operation to ensure that samples are collected and handled properly and to verify

the sampling site location. The field sampling staff then remove the target species

directly from the sampling device and ensure that sample collection, processing,

and preservation are conducted as prescribed in sample collection protocols, with

minimal chance of contamination. This is an excellent method of obtaining specimens of commercially important target species, particularly from the Great Lakes

and coastal estuarine areas (Versar, 1982). More detailed descriptions of active

sampling devices and their use are provided in Battelle (1975), Bennett, et al.,

(1970), Gunderson and Ellis (1986), Hayes (1983), Mearns and Allen (1978), Pitt

(1981), Puget Sound Estuary Program (1990b), Versar (1982), and Weber (1973).

6.2.1.2 Passive Collection&

Passive collection methods employ a wide array of sampling devices for fish and

shellfish, including gill nets, fyke nets, trammel nets, hoop nets, pound nets, and

d-traps. Passive collection methods generally require less fishing effort than

active methods but are usually less desirable for shallow water sample collection

because of the ability of many species to evade these entanglement and

entrapment devices. These methods normally yield a much greater catch than

would be required for a contaminant monitoring program and are time consuming

to deploy. In deep water, however, passive collection methods are generally

more efficient than active methods. Crawford and Luoma (1993) caution that

passive collection devices (e.g., gill nets) should be checked frequently to ensure

that captured fish do not deteriorate prior to removal from the sampling device.

Versar (1982, 1984) and Hubert (1983) describe passive sampling devices and

their use in more detail. It is highly desirable to collect live, intact fish that have

not been mutilated by the collection gear and that do not have any skin

lacerations or fin deterioration. For these reasons, EPA recommends that fish

captured in passive collection devices not remain in the water for more than

24 hours after the passive collection device is first deployed and that specimens

that show any skin or fin deterioration or external lacerations of any kind not used

for chemical analysis. 

Purchasing fish and shellfish from commercial fishers using passive collection

methods is acceptable; however, field sampling staff should accompany the

fishers during both the deployment and collection operations to ensure that

samples are collected and handled properly and to verify the sampling site

location. The field sampling staff can then ensure that sample collection,

processing, and preservation are conducted as prescribed in sample collection

protocols, with minimal chance of contamination.
6. FIELD PROCEDURES

6-45

6.2.2 Preservation of Sample Integrity

The primary QA consideration in sample collection, processing, preservation, and

shipping procedures is the preservation of sample integrity to ensure the accuracy

of target analyte analyses. Sample integrity is preserved by prevention of loss of

contaminants already present in the tissues and prevention of extraneous tissue

contamination (Smith, 1985).

Loss of contaminants already present in fish or shellfish tissues can be prevented

in the field by ensuring that the skin on fish specimens has not been lacerated by

the sampling gear or that the carapace of crustaceans or shells of bivalves have

not been cracked during sample collection resulting in loss of tissues and/or fluids

that may contain contaminants. Once the samples have reached the laboratory,

further care must be taken during thawing (if specimens are frozen) to ensure that

all liquids from the thawed specimens are retained with the tissue sample as

appropriate (see Sections 7.2.2, 7.2.3, and 7.2.4).

Sources of extraneous tissue contamination include contamination from sampling

gear, grease from ship winches or cables, spilled engine fuel (gasoline or diesel),

engine exhaust, dust, ice chests, and ice used for cooling. All potential sources

of contamination in the field should be identified and appropriate steps taken to

minimize or eliminate them. For example, during sampling, the boat should be

positioned so that engine exhausts do not fall on the deck. Ice chests should be

scrubbed clean with detergent and rinsed with distilled water after each use to

prevent contamination. To avoid contamination from melting ice, samples should

be placed in waterproof plastic bags (Stober, 1991). Sampling equipment that

has obviously been contaminated by oils, grease, diesel fuel, or gasoline should

not be used. All utensils or equipment that will be used directly in handling fish

or shellfish (e.g., fish measuring board or calipers) should be cleaned in the

laboratory prior to each sampling trip, rinsed in acetone and pesticide-grade

hexane, and stored in aluminum foil until use (Versar, 1982). Between sampling

sites, the field collection team should clean each measurement device by rinsing

it with ambient water and rewrapping it in aluminum foil to prevent contamination.

Note: Ideally, all sample processing (e.g., resections) should be performed at a

sample processing facility under cleanroom conditions to reduce the possibility of

sample contamination (Schmitt and Finger, 1987; Stober, 1991). However, there

may be some situations in which state staff find it necessary to fillet finfish or

resect edible turtle or shellfish tissues in the field prior to packaging the samples

for shipment to the processing laboratory. This practice should be avoided

whenever possible. If states find that filleting fish or resecting other edible tissues

must be performed in the field, a clean area should be set up away from sources

of diesel exhaust and areas where gasoline, diesel fuel, or grease are used to

help reduce the potential for surface and airborne contamination of the samples

from PAHs and other contaminants. Use of a mobile laboratory or use of a

portable resection table and enclosed hood would provide the best environment

for sample processing in the field. General guidance for conducting sample
6. FIELD PROCEDURES

6-46

processing under cleanroom conditions is provided in Section 7.2.1. States

should review this guidance to ensure that procedures as similar as possible to

those recommended for cleanroom processing are followed. If sample processing

is conducted in the field, a notation should be made in the field records and on the

sample processing record (see Figure 7-2). Procedures for laboratory processing

and resection are described in Section 7.2. Procedures for assessing sources of

sample contamination through the analyses of field and processing blanks are

described in Section 8.3.3.6.

6.2.3 Field Recordkeeping

Thorough documentation of all field sample collection and processing activities is

necessary for proper interpretation of field survey results. For fish and shellfish

contaminant studies, it is advisable to use preprinted waterproof data forms,

indelible ink, and writing implements that can function when wet (Puget Sound

Estuary Program, 1990b). When multicopy forms are required, no-carbonrequired (NCR) paper is recommended because it allows information to be

forwarded on the desired schedule and retained for the project file at the same

time.

Four separate preprinted sample tracking forms should be used for each sampling

site to document field activities from the time the sample is collected through

processing and preservation until the sample is delivered to the processing

laboratory. These are

 Field record form

 Sample identification label

 Chain-of-custody (COC) label or tag

 COC form.

6.2.3.1 Field Record Form&

The following information should be included on the field record for each sampling

site in both Tier 1 screening (Figures 6-3 and 6-4) and Tier 2 intensive studies as

appropriate (Figures 6-5 and 6-6):

 Project number

 Sampling date and time (give date in a Year 2000 compliant format

[YYYYMMDD] and specify convention used for time, e.g., 24-h clock)

 Sampling site location (including site name and number, county/parish,

latitude/longitude, waterbody name/segment number, waterbody type, and site

description)

 Sampling depth (specify units of depth)

 Collection method

 Collectors' names and signatures

 Agency (including telephone number and address)
6. FIELD PROCEDURES

6-47

Figure 6-3. Example of a field record for fish contaminant monitoring

program—screening study.
6. FIELD PROCEDURES

6-48

Figure 6-4. Example of a field record for shellfish contaminant monitoring

program—screening study.
6. FIELD PROCEDURES

6-49

Figure 6-5. Example of a field record for fish contaminant monitoring

program—intensive study.
6. FIELD PROCEDURES

6-50

Figure 6-5. (continued)
6. FIELD PROCEDURES

6-51

Figure 6-6. Example of a field record for shellfish contaminant monitoring

program—intensive study.
6. FIELD PROCEDURES

6-52

Species Name or Code Sample Type

Total Length or Size (mm) Sampling Site (name/number)

Specimen Number Sampling Date (YYYMMDD)

Time (24-h clock)

Figure 6-7. Example of a sample identification label.

 Species collected (including species common and scientific name, composite

sample number, individual specimen number, number of individuals per

composite sample, number of replicate samples, total length/size [mm], sex

[male, female, indeterminate]) 

Note: States should specify a unique numbering system to track samples for their

own fish and shellfish contaminant monitoring programs.

 Percent difference in size between the smallest and largest specimens to be

composited (smallest individual length [or size] divided by the largest

individual length [or size] x 100; should be >75 percent) and mean composite

length or size (mm)

 Notes (including visible morphological abnormalities, e.g., fin erosion, skin

ulcers, cataracts, skeletal and exoskeletal anomalies, neoplasms, or

parasites).

6.2.3.2 Sample Identification Label&

A sample identification label should be completed in indelible ink for each

individual fish or shellfish specimen after it is processed to identify each sample

uniquely (Figure 6-7). The following information should be included on the sample

identification label:

 Species scientific name or code number

 Total length/size of specimen (mm)

 Specimen number

 Sample type: F (fish fillet analysis only)

S (shellfish edible portion analysis only)

W (whole fish analysis)

O (other fish tissue analysis)
6. FIELD PROCEDURES

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Project Number Collection Agency (name, address, phone)

Sampling Site (name and/or ID number) Sampler (name and signature)

Composition Number/Specimen Number(s) Chemical Analyses

* All target analytes

* Others (specify) 

Study Type

Sampling Date (YYYYMMDD) Time (24-h clock) Screening Intensive

Phase I *

Phase II *

Species Name or Code Processing Type of Ice

Whole Body Resection Wet Dry

Comments

Figure 6-8. Example of a chain-of-custody tag or label.

 Sampling site&waterbody name and/or identification number

 Sampling date/time (give date in a Year 2000 compliant format [YYYYMMDD]

and specify convention for time, e.g., 24-h clock).

A completed sample identification label should be taped to each aluminum-foilwrapped specimen and the specimen should be placed in a waterproof plastic

bag.

6.2.3.3 Chain-of-Custody Label or Tag&

A COC label or tag should be completed in indelible ink for each individual fish

specimen. The information to be completed for each fish is shown in Figure 6-8.

After all information has been completed, the COC label or tag should be taped

or attached with string to the outside of the waterproof plastic bag containing the

individual fish sample. Information on the COC label/tag should also be recorded

on the COC form (Figure 6-9).

Because of the generally smaller size of shellfish, several individual aluminum-foilwrapped shellfish specimens (within the same composite sample) may be placed

in the same waterproof plastic bag. A COC label or tag should be completed in

indelible ink for each shellfish composite sample. If more than 10 individual
6. FIELD PROCEDURES

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Figure 6-9. Example of a chain-of-custody record form.
6. FIELD PROCEDURES

6-55

shellfish are to be composited, several waterproof plastic bags may have to be

used for the same composite. It is important not to place too many individual 

specimens in the same plastic bag to ensure proper preservation during shipping,

particularly during summer months. Information on the COC label/tag should also

be recorded on the COC form (Figure 6-9).

6.2.3.4 Chain-of-Custody Form&

A COC form should be completed in indelible ink for each shipping container (e.g.,

ice chest) used. Information recommended for documentation on the COC form

(Figure 6-9) is necessary to track all samples from field collection to receipt at the

processing laboratory. In addition, this form can be used for tracking samples

through initial laboratory processing (e.g., resection) as described in Section 7.2.

Prior to sealing the ice chest, one copy of the COC form and a copy of the field

record sheet should be sealed in a resealable waterproof plastic bag. This plastic

bag should be taped to the inside cover of the ice chest so that it is maintained

with the samples being tracked. Ice chests should be sealed with reinforced tape

for shipment.

6.2.3.5 Field Logbook&

In addition to the four sample tracking forms discussed above, the field collection

team should document in a field logbook any additional information on sample

collection activities, hydrologic conditions (e.g., tidal stage), weather conditions,

boat or equipment operations, or any other unusual activities observed (e.g.,

dredging) or problems encountered that would be useful to the program manager

in evaluating the quality of the fish and shellfish contaminant monitoring data.

6.3 SAMPLE HANDLING

6.3.1 Sample Selection

6.3.1.1 Species Identification&

As soon as fish, shellfish, and turtles are removed from the collection device, they

should be identified by species. Nontarget species or specimens of target species

that do not meet size requirements (e.g., juveniles) should be returned to the

water. Species identification should be conducted only by experienced personnel

knowledgeable of the taxonomy of species in the waterbodies included in the

contaminant monitoring program. Taxonomic keys, appropriate for the waters

being sampled, should be consulted for species identification. Because the

objective of both the screening and intensive monitoring studies is to determine

the magnitude of contamination in specific fish, shellfish, and turtle species, it is

necessary that all individuals used in a composite sample be of a single species.

Note: Correct species identification is important and different species should

never be combined in a single composite sample.
6. FIELD PROCEDURES

6-56

When sufficient numbers of the target species have been identified to make up a

composite sample, the species name and all other appropriate information should

be recorded on the field record forms (Figures 6-3 through 6-6).

Note: EPA recommends that, when turtles are used as the target species, target

analyte concentrations be determined for each turtle rather than for a composite

turtle sample.

6.3.1.2 Initial Inspection and Sorting&

Individual fish of the selected target species should be rinsed in ambient water to

remove any foreign material from the external surface. Large fish should be

stunned by a sharp blow to the base of the skull with a wooden club or metal rod.

This club or rod should be used solely for the purpose of stunning fish, and care

should be taken to keep it reasonably clean to prevent contamination of the

samples (Versar, 1982). Small fish may be placed on ice immediately after

capture to stun them, thereby facilitating processing and packaging procedures.

Once stunned, individual specimens of the target species should be grouped by

species and general size class and placed in clean holding trays to prevent

contamination. All fish should be inspected carefully to ensure that their skin and

fins have not been damaged by the sampling equipment, and damaged specimens should be discarded (Versar, 1982).

Freshwater turtles should be rinsed in ambient water and their external surface

scrubbed if necessary to remove any foreign matter from their carapace and

limbs. Each turtle should be inspected carefully to ensure that the carapace and

extremities have not been damaged by the sampling equipment, and damaged

specimens should be discarded (Versar, 1982). Care should be taken when

handling large turtles, particularly snapping turtles; many can deliver severe bites.

Particularly during procedures that place fingers or hands within striking range of

the sharp jaws, covering the turtle's head, neck, and forelimbs with a cloth towel

or sack and taping it in place is often sufficient to prevent injury to the field

sampling crew (Frye, 1994).

After inspection, each turtle should be placed individually in a heavy burlap sack

or canvas bag tied tightly with a strong cord and then placed in an ice-filled cooler.

Placing turtles on ice will slow their metabolic rate, making them easier to handle.

Note: It is recommended that each turtle be analyzed as an individual sample,

especially if the target turtle species is not abundant in the waterbody being

sampled or if the collected individuals differ greatly in size or age. Analysis of

individual turtles can provide an estimate of the maximum contaminant

concentrations to which recreational or substistence fishers are exposed. Target

analyte concentrations in composite samples represent averages for a specific

target species population. The use of these values in risk assessment is

appropriate if the objective is to estimate the average concentration to which

consumers of the target species are exposed over a long period of time. The use

of long exposure periods (e.g., 70 years) is typical for the assessment of
6. FIELD PROCEDURES

6-57

carcinogenic effects, which may be manifest over an entire lifetime (see Volume

II of this guidance series). Noncarcinogenic effects, on the other hand, may cause

acute health effects over a relatively short period of time (e.g., hours or days) after

consumption. The maximum target analyte contaminant concentration may be

more appropriate than the average target analyte concentration for use with

noncarginogenic target analytes (U.S. EPA, 1989d). This is especially important

for those target analytes for which acute exposures to very high concentrations

may be toxic to consumers. 

Stone et al. (1980) reported extremely high concentrations of PCBs in various

tissues of snapping turtles from a highly contaminated site on the Hudson River.

Contaminant analysis of various turtle tissues showed mean PCB levels of 2,991

ppm in fatty tissue, 66 ppm in liver tissue, and 29 ppm in eggs as compared to 4

ppm in skeletal muscle. Clearly, inclusion of the fatty tissue, liver, and eggs with

the muscle tissues as part of the edible tissues will increase observed residue

concentrations over those detected in muscle tissue only. States interested in

using turtles as target species should review Appendix C for additional information

on the use of individual samples in contaminant monitoring programs.

Bivalves (oysters, clams, scallops, and mussels) adhering to one another should

be separated and scrubbed with a nylon or natural fiber brush to remove any

adhering detritus or fouling organisms from the exterior shell surfaces (NOAA,

1987). All bivalves should be inspected carefully to ensure that the shells have

not been cracked or damaged by the sampling equipment and damaged

specimens should be discarded (Versar, 1982). Crustaceans, including shrimp,

crabs, crayfish, and lobsters, should be inspected to ensure that their

exoskeletons have not been cracked or damaged during the sampling process,

and damaged specimens should be discarded (Versar, 1982). After shellfish have

been rinsed, individual specimens should be grouped by target species and

placed in clean holding trays to prevent contamination.

A few shellfish specimens may be resected (edible portions removed) to determine wet weight of the edible portions. This will provide an estimate of the

number of individuals required to ensure that the recommended sample weight

(200 g) is attained. Note: Individuals used to determine the wet weight of the

edible portion should not be used for target analyte analyses.

6.3.1.3 Length or Size Measurements&

Each fish within the selected target species should be measured to determine

total body length (mm). To be consistent with the convention used by most

fisheries biologists in the United States, maximum body length should be

measured as shown in Figure 6-10. The maximum body length is defined as the

length from the anterior-most part of the fish to the tip of the longest caudal fin ray

(when the lobes of the caudal fin are compressed dorsoventrally) (Anderson and

Gutreuter, 1983). 
6. FIELD PROCEDURES

6-58

a Maximum body length is the length from the anterior-most part of the fish to the tip of the

longest caudal fin ray (when the lobes of the caudal fin are compressed dorsoventrally

(Anderson and Gutreuter, 1983).

b Carapace width is the lateral distance across the carapace (from tip of spine to tip of spine

(U.S. EPA, 1990c). c Height is the distance from the umbo to the anterior (ventral) shell margin (Galtsoff, 1964).

d Body length is the distance from the tip of the rostrum to the tip of the telson (Texas Water

Commission, 1990). e Carapace length is distance from top of rostrum to the posterior margin of the carapace.

Figure 6-10. Recommended measurements of body length and size for fish, 

shellfish, and turtles.
6. FIELD PROCEDURES

6-59

e Carapace length is the distance from the anterior-most edge of the groove between the

horns directly above the eyes, to the rear edge of the top part of the carapace as measured

along the middorsal line of the back (Laws of Florida Chapter 46-24.003).

f Tail length is the distance measured lengthwise along the top middorsal line of the entire tail

to rear-most extremity (this measurement shall be conducted with the tail in a flat straight

position with the tip of the tail closed) (Laws of Florida Chapter 46-24.003).

g Carapace length is the distance from the rear of the eye socket to the posterior margin of

the carapace (New York Environmental Conservation Law 13-0329.5.a and Massachusetts

General Laws Chapter 130).

h Carapace length is the straight-line distance from the anterior margin to the posterior margin

of the shell (Conant and Collins, 1991).

Figure 6-10. (continued)
6. FIELD PROCEDURES

6-60

Each turtle within the selected target species should be measured to determine

total carapace length (mm). To be consistent with the convention used by most

herpetologists in the United States, carapace length should be measured as

shown in Figure 6-10. The maximum carapace length is defined as the straight

line distance from the anterior edge of the carapace to the posterior edge of the

carapace (Conant and Collins, 1991).

For shellfish, each individual specimen should be measured to determine the

appropriate body size (mm). As shown in Figure 6-9, the recommended body

measurements differ depending on the type of shellfish being collected. Height

is a standard measurement of size for oysters, mussels, clams, scallops, and

other bivalve molluscs (Abbott, 1974; Galtsoff, 1964). The height is the distance

from the umbo to the anterior (ventral) shell margin. For crabs, the lateral width

of the carapace is a standard size measurement (U.S. EPA, 1990c); for shrimp

and crayfish, the standard measurement of body size is the length from the rostrum to the tip of the telson (Texas Water Commission, 1990); and for lobsters,

two standard measurements of body size are commonly used. For clawed and

spiny lobsters, the standard size is the length of the carapace. For spiny lobsters,

the length of the tail is also used as a standard size measurement.

6.3.1.4 Sex Determination (Optional)&

An experienced fisheries biologist can often make a preliminary sex determination

for fish by visual inspection. The body of the fish should not be dissected in the

field to determine sex; sex can be determined through internal examination of the

gonads during laboratory processing (Section 7.2.2.4).

An experienced herpetologist can often make a preliminary sex determination of

a turtle by visual inspection in the field. The plastron (ventral portion of the

carapace) is usually flatter in the female and the tail is less well developed than

in the male. The plastron also tends to be more concave in the male (Holmes,

1984). For the common snapping turtle (Chelydra serpentina), the cloaca of the

female is usually located inside or at the perimeter of the carapace, while the

cloaca of the male extends slightly beyond the perimeter of the carapace. The

carapace of the turtle should never be resected in the field to determine sex; sex

can be determined through internal examination of the gonads during laboratory

processing (Section 7.2.3.4.). For shellfish, a preliminary sex determination can

be made by visual inspection only for crustaceans. Sex cannot be determined in

bivalve molluscs without shucking the bivalves and microscopically examining

gonadal material. Bivalves should not be shucked in the field to determine sex;

sex determination through examination of the gonads can be performed during

laboratory processing if desired (Section 7.2.4.2).

6.3.1.5 Morphological Abnormalities (Optional)&

If resources allow, states may wish to consider documenting external gross

morphological conditions in fish from contaminated waters. Severely polluted
6. FIELD PROCEDURES

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aquatic habitats have been shown to produce a higher frequency of gross

pathological disorders than similar, less polluted habitats (Krahn et al., 1986;

Malins et al., 1984, 1985; Mix, 1986; Sinderman, 1983; and Sinderman et al.,

1980).

Sinderman et al. (1980) reviewed the literature on the relationship of fish

pathology to pollution in marine and estuarine environments and identified four

gross morphological conditions acceptable for use in monitoring programs:

 Fin erosion

 Skin ulcers

 Skeletal anomalies

 Neoplasms (i.e., tumors).

Fin erosion is the most frequently observed gross morphological abnormality in

polluted areas and is found in a variety of fishes (Sinderman, 1983). In demersal

fishes, the dorsal and anal fins are most frequently affected; in pelagic fishes, the

caudal fin is primarily affected.

Skin ulcers have been found in a variety of fishes from polluted waters and are the

second most frequently reported gross abnormality. Prevalence of ulcers

generally varies with season and is often associated with organic enrichment

(Sinderman, 1983).

Skeletal anomalies include abnormalities of the head, fins, gills, and spinal column

(Sinderman, 1983). Skeletal anomalies of the spinal column include fusions,

flexures, and vertebral compressions.

Neoplasms or tumors have been found at a higher frequency in a variety of

polluted areas throughout the world. The most frequently reported visible tumors

are liver tumors, skin tumors (i.e., epidermal papillomas and/or carcinomas), and

neurilemmomas (Sinderman, 1983).

The occurrence of fish parasites and other gross morphological abnormalities that

are found at a specific site should be noted on the field record form. States

interested in documenting morphological abnormalities in fish should review the

protocols for fish pathology studies recommended in the Puget Sound Estuary

Program (1990c) and those described by Goede and Barton (1990).

6.3.2 Sample Packaging

6.3.2.1 Fish&

After initial processing to determine species, size, sex, and morphological

abnormalities, each fish should be individually wrapped in extra heavy duty

aluminum foil. Spines on fish should be sheared to minimize punctures in the

aluminum foil packaging (Stober, 1991). The sample identification label shown

in Figure 6-7 should be taped to the outside of each aluminum foil package, each

individual fish should be placed into a waterproof plastic bag and sealed, and the
6. FIELD PROCEDURES

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COC tag or label should be attached to the outside of the plastic bag with string

or tape. All of the packaged individual specimens in a composite sample should

be kept together (if possible) in one large waterproof plastic bag in the same

shipping container (ice chest) for transport. Once packaged, samples should be

cooled on ice immediately.

6.3.2.2 Turtles&

After inital processing to determine the species, size (carapace length), and sex,

each turtle should be placed on ice in a separate burlap or canvas bag and stored

on ice for transport to the processing laboratory. A completed sample identification label (Figure 6-7) should be attached with string around the neck or one of the

turtle's extremities and the COC tag or label should be attached to the outside of

the bag with string or tape. Note: Bagging each turtle should not be undertaken

until the specimen has been sufficiently cooled to induce a mild state of torpor,

thus facilitating processing. The samplers should work rapidly to return each

turtle to the ice chest as soon as possible after packaging as the turtle may

suddenly awaken as it warms thus becoming a danger to samplers (Frye, 1994).

As mentioned in Section 6.3.1, states should analyze turtles individually rather

than compositing samples. This is especially important when very few specimens

are collected at a sampling site or when specimens of widely varying size or age

are collected.

Note: When a large number of individual specimens in the same composite

sample are shipped together in the same waterproof plastic bag, the samples

must have adequate space in the bag to ensure that contact with ice can occur,

thus ensuring proper preservation during shipping. This is especially important

when samples are collected during hot weather and/or when the time between

field collection and delivery to the processing laboratory approaches the maximum

shipping time (Table 6-8).

6.3.2.3 Shellfish&

After initial processing to determine species, size, sex, and morphological

abnormalities, each shellfish specimen should be wrapped individually in extra

heavy duty aluminum foil. A completed sample identification label (Figure 6-7)

should be taped to the outside of each aluminum foil package. Note: Some

crustacean species (e.g., blue crabs and spiny lobsters) have sharp spines on

their carapace that might puncture the aluminum foil wrapping. Carapace spines

should never be sheared off because this would destroy the integrity of the

carapace. For such species, one of the following procedures should be used to

reduce punctures to the outer foil wrapping:

 Double-wrap the entire specimen in extra heavy duty aluminum foil.

 Place clean cork stoppers over the protruding spines prior to wrapping the

specimen in aluminum foil. 
6. FIELD PROCEDURES

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Table 6-8. Recommendations for Preservation of Fish, Shellfish, and Turtle Samples

from Time of Collection to Delivery at the Processing Laboratory

Sample type

Number per

composite Container Preservation

Maximum

shipping

time

Fisha

Whole fish

(to be filleted)

3-10 Extra heavy duty

aluminum foil wrap of

each fish.b

 Each fish is

placed in a waterproof

plastic bag.

Cool on wet ice or blue

ice packets

(preferred method)

 or

Freeze on dry ice

only if shipping 

time will exceed 24

hours

24 hours

48 hours

Whole fish 3-10 Same as above. Cool on wet ice or blue

ice packets

 or

Freeze on dry ice

24 hours

48 hours

Shellfisha

Whole shellfish

(to be resected for

edible tissue)

3-50c Extra heavy duty

aluminum foil wrap of

each specimen.b

Shellfish in the same

composite sample may

be placed in the same

waterproof plastic bag.

Cool on wet ice or blue

ice packets

(preferred method)

 or

Freeze on dry ice

if shipping time

will exceed 24 hours

24 hours

48 hours

Whole shellfish 3-50c Same as above. Cool on wet ice or blue

ice packets

 or

Freeze on dry ice

24 hours

48 hours

Whole turtles

(to be resected for

edible tissue)

1d Heavy burlap or

canvas bags.

Cool on wet ice or blue

ice packets (preferred

method)

 or

Freeze on dry ice if

shipping time to exceed

24 hours

24 hours

48 hours

a Use only individuals that have attained at least legal harvestable or consumable size. b Aluminum foil should not be used for long-term storage of any sample (i.e., whole organisms, fillets, or

homogenates) that will be analyzed for metals. c Species and size dependent. For very small shellfish species, more than 50 individuals may be required to

achieve the 200-g composite sample mass recommended for screening studies. d Turtles should be analyzed as individual rather than as composite samples.

 Wrap the spines with multiple layers of foil before wrapping the entire specimen in aluminum foil.

All of the individual aluminum-foil-wrapped shellfish specimens (in the same

composite sample) should be placed in the same waterproof plastic bag for

transport. In this case, a COC tag or label should be completed for the composite

sample and appropriate information recorded on the field record sheet and COC

form. The COC label or tag should then be attached to the outside of the plastic
6. FIELD PROCEDURES

6-64

bag with string or tape. For composite samples containing more than 10 shellfish

specimens or especially large individuals, additional waterproof plastic bags may

be required to ensure proper preservation. Once packaged, composite samples

should be cooled on ice immediately. Note: When a large number of individual

specimens in the same composite sample are shipped together in the same

waterproof plastic bag, the samples must have adequate space in the bag to

ensure that contact with ice can occur; thus ensuring proper preservation during

shipping. This is especially important when samples are collected during hot

weather and/or when the time between field collection and delivery to the

processing laboratory approaches the maximum shipping time (Table 6-8).

6.3.3 Sample Preservation

The type of ice to be used for shipping should be determined by the length of time

the samples will be in transit to the processing laboratory and the sample type to

be analyzed (Table 6-8).

6.3.3.1 Fish, Turtles, or Shellfish To Be Resected&

Note: Ideally fish, turtles, and shellfish specimens should not be frozen prior to

resection if analyses will include edible tissue only because freezing may cause

some internal organs to rupture and contaminate fillets or other edible tissues

(Stober, 1991; U.S. EPA, 1986b). Wet ice or blue ice (sealed prefrozen ice

packets) is recommended as the preservative of choice when the fish fillet, turtle

meat, or shellfish edible portions are the primary tissues to be analyzed. Samples

shipped on wet or blue ice should be delivered to the processing laboratory within

24 hours (Smith, 1985; U.S. EPA, 1990d). If the shipping time to the processing

laboratory will exceed 24 hours, dry ice should be used.

Note: One exception to the use of dry ice for long-term storage is if fish or

shellfish are collected as part of extended offshore field surveys. States involved

in these types of field surveys may employ shipboard freezers to preserve

samples for extended periods rather than using dry ice. Ideally, all fish should be

resected in cleanrooms aboard ship prior to freezing.

6.3.3.2 Fish, Turtles, or Shellfish for Whole-Body Analysis&

At some sites, states may deem it necessary to collect fish, turtles, or shellfish for

whole-body analysis if a local subpopulation of concern typically consumes whole

fish, turtles, or shellfish. If whole fish, turtles, or shellfish samples are to be

analyzed, either wet ice, blue ice, or dry ice may be used; however, if the shipping

time to the processing laboratory will exceed 24 hours, dry ice should be used.

Dry ice requires special packaging precautions before shipping by aircraft to

comply with U.S. Department of Transportation (DOT) regulations. The Code of

Federal Regulations (49 CFR 173.217) classifies dry ice as Hazard Class 9

UN1845 (Hazardous Material). These regulations specify the amount of dry ice
6. FIELD PROCEDURES

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that may be shipped by air transport and the type of packaging required. For

each shipment by air exceeding 5 pounds of dry ice per package, advance

arrangements must be made with the carrier. Not more than 441 pounds of dry

ice may be transported in any one cargo compartment on any aircraft unless the

shipper has made special written arrangements with the aircraft operator.

The regulations further specify that the packaging must be designed and

constructed to permit the release of carbon dioxide gas to prevent a buildup of

pressure that could rupture the package. If samples are transported in a cooler,

several vent holes should be drilled to allow carbon dioxide gas to escape. The

vents should be near the top of the vertical sides of the cooler, rather than in the

cover, to prevent debris from falling into the cooler. Wire screen or cheesecloth

should be installed in the vents to keep foreign materials from contaminating the

cooler. When the samples are packaged, care should be taken to keep these

vents open to prevent the buildup of pressure.

Dry ice is exempted from shipping certification requirements if the amount is less

than 441 pounds and the package meets design requirements. The package

must be marked "Carbon Dioxide, Solid" or "Dry Ice" with a statement indicating

that the material being refrigerated is to be used for diagnostic or treatment

purposes (e.g., frozen tissue samples).

6.3.4 Sample Shipping

The fish, turtle, and shellfish samples should be hand-delivered or shipped to the

processing laboratory as soon as possible after collection. The time the samples

were collected and time of their arrival at the processing laboratory should be

recorded on the COC form (Figure 6-9).

If the sample is to be shipped rather than hand-delivered to the processing

laboratory, field collection staff must ensure the samples are packed properly with

adequate ice layered between samples so that sample degradation does not

occur. In addition, a member of the field collection staff should telephone ahead

to the processing laboratory to alert them to the anticipated delivery time of the

samples and the name and address of the carrier to be used. Field collection staff

should avoid shipping samples for weekend delivery to the processing laboratory

unless prior plans for such a delivery have been agreed upon with the processing

laboratory staff.
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-1

SECTION 7

LABORATORY PROCEDURES I — SAMPLE HANDLING

This section provides guidance on laboratory procedures for sample receipt,

chain-of-custody, processing, distribution, analysis, and archiving. Planning,

documentation, and quality assurance and quality control of all laboratory

activities are emphasized to ensure that (1) sample integrity is preserved during

all phases of sample handling and analysis, (2) chemical analyses are performed

cost-effectively and meet program data quality objectives, and (3) data produced

by different states and regions are comparable.

Laboratory procedures should be documented in a Work/QA Project Plan (U.S.

EPA, 1980b) as described in Appendix I. Routine sample processing and analysis

procedures should be prepared as standard operating procedures (SOPs) (U.S.

EPA, 1984b).

7.1 SAMPLE RECEIPT AND CHAIN-OF-CUSTODY

Fish, shellfish, and turtle samples may be shipped or hand-carried from the field

according to one or more of the following pathways:

• From the field to a state laboratory for sample processing and analysis

• From the field to a state laboratory for sample processing and shipment of

composite sample aliquots to a contract laboratory for analysis

• From the field to a contract laboratory for sample processing and analysis.

Sample processing and distribution for analysis ideally should be performed by

one processing laboratory. Transportation of samples from the field should be

coordinated by the sampling team supervisor and the laboratory supervisor

responsible for sample processing and distribution (see Section 6.3.4). An

accurate written custody record must be maintained so that possession and

treatment of each sample can be traced from the time of collection through

analysis and final disposition.

Fish, shellfish, and turtle samples should be brought or shipped to the sample

processing laboratory in sealed containers accompanied by a copy of the sample

request form (Figure 6-1), a chain-of-custody form (Figure 6-9), and the field

records (Figures 6-3 through 6-6). Each time custody of a sample or set of

samples is transferred, the Personnel Custody Record of the COC form must be

completed and signed by both parties. Corrections to the COC form should be

made in indelible ink by drawing a single line through the original entry, entering
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-2

the correct information and the reason for the change, and initialing and dating the

correction. The original entry should never be obscured.

When custody is transferred from the field to the sample processing laboratory,

the following procedure should be used:

• Note the shipping time. If samples have been shipped on wet or blue ice,

check that the shipping time has not exceeded 24 hours.

• Check that each shipping container has arrived undamaged and that the seal

is intact.

• Open each shipping container and remove the copy of the sample request

form, the COC form, and the field records.

• Note the general condition of the shipping container (samples iced properly

with no leaks, etc.) and the accompanying documentation (dry, legible, etc.).

• Locate individuals in each composite sample listed on the COC form and note

the condition of their packaging. Individual specimens should be properly

wrapped and labeled. Note any problems (container punctured, illegible

labels, etc.) on the COC form.

• If individuals in a composite are packaged together, check the contents of

each composite sample container against the field record for that sample to

ensure that the individual specimens are properly wrapped and labeled. Note

any discrepancies or missing information on the COC form.

• Initial the COC form and record the date and time of sample receipt.

• Enter the following information for each composite sample into a permanent

laboratory record book and, if applicable, a computer database:

— Sample identification number (specify conventions for the composite

sample number and the specimen number) Note: EPA recommends

processing and analysis of turtles as individual samples.

— Receipt date (use Year 2000 comliant format [YYYYMMDD])

— Sampling date (use Year 2000 comliant format [YYYYMMDD])

— Sampling site (name and/or identification number)

— Fish, turtle, and shellfish species (scientific name or code number)

— Total length of each fish, carapace length of each turtle, or size of each

shellfish (mm)
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-3

• If samples have been shipped on wet or blue ice, distribute them immediately

to the technician responsible for resection (see Section 7.2). See

Section 7.2.3 for the procedure for processing turtle samples as individual

samples. If samples have been shipped on dry ice, they may be distributed

immediately to the technician for processing or stored in a freezer at -20 C

for later processing. Once processed, fillets or edible portions of fish, turtles,

or shellfish or tissue homogenates, should be stored according to the

procedures described in Section 7.2 and in Table 7-1. Note: Holding times

in Table 7-1 are maximum times recommended for holding samples from the

time they are received at the laboratory until they are analyzed. These

holding times are based on guidance that is sometimes administrative rather

than technical in nature; there are no promulgated holding time criteria for

tissues (U.S. EPA, 1995i). If states choose to use longer holding times, they

must demonstrate and document the stability of the target analyte residues

over the extended holding times. 

7.2 SAMPLE PROCESSING

This section includes recommended procedures for preparing composite

homogenate samples of fish fillets and edible portions of shellfish and individual

samples of edible portions of freshwater turtles as required in screening and

intensive studies. Recommended procedures for preparing whole fish composite

homogenates are included in Appendix J for use by states in assessing the

potential risk to local subpopulations known to consume whole fish or shellfish.

7.2.1 General Considerations

All laboratory personnel performing sample processing procedures (see

Sections 7.2.2, 7.2.3, and 7.2.4) should be trained or supervised by an

experienced fisheries biologist. Care must be taken during sample processing to

avoid contaminating samples. Schmitt and Finger (1987) have demonstrated that

contamination of fish flesh samples is likely unless the most exacting clean

dissection procedures are used. Potential sources of contamination include dust,

instruments, utensils, work surfaces, and containers that may contact the

samples. All sample processing (i.e., filleting, removal of other edible tissue,

homogenizing, compositing) should be done in an appropriate laboratory facility

under cleanroom conditions (Stober, 1991). Cleanrooms or work areas should be

free of metals and organic contaminants. Ideally, these areas should be under

positive pressure with filtered air (HEPA filter class 100) (California Department

of Fish and Game, 1990). Periodic wipe tests should be conducted in clean areas

to verify the absence of significant levels of metal and organic contaminants. All

instruments, work surfaces, and containers used to process samples must be of

materials that can be cleaned easily and that are not themselves potential sources

of contamination. More detailed guidance on establishing trace metal cleanrooms

is provided in U.S. EPA (1995a).
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-4

Table 7-1. Recommendations for Container Materials, Preservation, and Holding

Times for Fish, Shellfish, and Turtle Tissues from Receipt at Sample 

Processing Laboratory to Analysis

Analyte Matrix Sample container

Storage

Preservation Holding timea

Mercury Tissue (fillets and edible

portions, homogenates)

Plastic, borosilicate

glass, quartz, PTFE

Freeze at <-20 C 28 daysb

Other metals Tissue (fillets and edible

portions, homogenates)

Plastic, borosilicate

glass, quartz, PTFE

Freeze at <-20 C 6 monthsc

Organics Tissue (fillets and edible

portions, homogenates)

Borosilicate glass,

PTFE, quartz,

aluminum foil

Freeze at <-20 C 1 yeard

Metals and

organics

Tissue (fillets and edible

portions, homogenates)

Borosilicate glass,

quartz, PTFE

Freeze at <-20 C 28 days

 (for mercury);

6 months 

(for other

metals); and 1

year (for

organics)

Lipids Tissue (fillets and edible

portions, homogenates)

Plastic, borosilicate

glass, quartz, PTFE

Freeze at <-20 C 1 year

PTFE = Polytetrafluoroethylene (Teflon).

a Maximum holding times recommended by EPA (1995i). b This maximum holding time is also recommended by the Puget Sound Estuary Program (1990e). The

California Department of Fish and Game (1990) and the USGS National Water Quality Assessment Program

(Crawford and Luoma, 1993) recommend a maximum holding time of 6 months for all metals, including

mercury. c This maximum holding time is also recommended by the California Department of Fish and Game (1990),

the 301(h) monitoring program (U.S. EPA, 1986b), and the USGS National Water Quality Assessment

Program (Crawford and Luoma, 1993). The Puget Sound Estuary Program (1990e) recommends a

maximum holding time of 2 years. d This maximum holding time is also recommended by the Puget Sound Estuary Program (1990e). The

California Department of Fish and Game (1990) and the USGS National Water Quality Assessment Program

(Crawford and Luoma, 1993) recommend a more conservative maximum holding time of 6 months. U.S.

EPA (1995b) recommends a maximum holding time of 1 year at -10 C for dioxins/furans.

To avoid cross-contamination, all equipment used in sample processing (i.e.,

resecting, homogenizing, and compositing) should be cleaned thoroughly before

each composite sample is prepared. Verification of the efficacy of cleaning

procedures should be documented through the analysis of processing blanks or

rinsates (see Section 8.3.3.6).

Because sources of organic and metal contaminants differ, it is recommended

that duplicate samples be collected, if time and funding permit, when analyses of

both organics and metals are required (e.g., for screening studies). One sample

can then be processed and analyzed for organics and the other can be processed

independently and analyzed for metals (Batelle, 1989; California Department of

Fish and Game, 1990; Puget Sound Estuary Program, 1990c, 1990d). If fish are

of adequate size, separate composites of individual fillets may be prepared and
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-5

analyzed independently for metals and organics. If only one composite sample

is prepared for the analyses of metals and organics, the processing equipment

must be chosen and cleaned carefully to avoid contamination by both organics

and metals. 

Suggested sample processing equipment and cleaning procedures by analysis

type are discussed in Sections 7.2.1.1 through 7.2.1.3. Other procedures may be

used if it can be demonstrated, through the analysis of appropriate blanks, that

no contamination is introduced (see Section 8.3.3.6).

7.2.1.1 Samples for Organics Analysis—

Equipment used in processing samples for organics analysis should be of

stainless steel, anodized aluminum, borosilicate glass, polytetrafluoroethylene

(PTFE), ceramic, or quartz. Polypropylene and polyethylene (plastic) surfaces,

implements, gloves, and containers are a potential source of contamination by

organics and should not be used. If a laboratory chooses to use these materials,

there should be clear documentation that they are not a source of contamination.

Filleting should be done on glass or PTFE cutting boards that are cleaned

properly between fish or on cutting boards covered with heavy duty aluminum foil

that is changed after each filleting. Tissue should be removed with clean, highquality, corrosion-resistant stainless steel or quartz instruments or with knives with

titanium blades and PTFE handles (Lowenstein and Young, 1986). Fillets or

tissue homogenates may be stored in borosilicate glass, quartz, or PTFE

containers with PTFE-lined lids or in heavy duty aluminum foil (see Table 7-1).

Prior to preparing each composite sample, utensils and containers should be

washed with detergent solution, rinsed with tap water, soaked in pesticide-grade

isopropanol or acetone, and rinsed with organic-free, distilled, deionized water.

Work surfaces should be cleaned with pesticide-grade isopropanol or acetone,

washed with distilled water, and allowed to dry completely. Knives, fish scalers,

measurement boards, etc., should be cleaned with pesticide-grade isopropanol

or acetone followed by a rinse with contaminant-free distilled water between each

fish sample (Stober, 1991).

7.2.1.2 Samples for Metals Analysis—

Equipment used in processing samples for metals analyses should be of quartz,

PTFE, ceramic, polypropylene, or polyethylene. The predominant metal

contaminants from stainless steel are chromium and nickel. If these metals are

not of concern, the use of high-quality, corrosion-resistant stainless steel for

sample processing equipment is acceptable. Quartz utensils are ideal but

expensive. For bench liners and bottles, borosilicate glass is preferred over

plastic (Stober, 1991). Knives with titanium blades and PTFE handles are

recommended for performing tissue resections (Lowenstein and Young, 1986).

Borosilicate glass bench liners are recommended. Filleting may be done on glass

or PTFE cutting boards that are cleaned properly between fish or on cutting
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-6

boards covered with heavy duty aluminum foil that is changed after each fish. 

Fillets or tissue homogenates may be stored in plastic, borosilicate glass, quartz,

or PTFE containers (see Table 7-1).

Prior to preparing each composite sample, utensils and containers should be

cleaned thoroughly with a detergent solution, rinsed with tap water, soaked in

acid, and then rinsed with metal-free water. Quartz, PTFE, glass, or plastic

containers should be soaked in 50 percent HN03, for 12 to 24 hours at room

temperature. Note: Chromic acid should not be used for cleaning any materials.

Acids used should be at least reagent grade. Stainless steel parts may be

cleaned as stated for glass or plastic, omitting the acid soaking step (Stober,

1991).

7.2.1.3 Samples for Both Organics and Metals Analyses—

As noted above, several established monitoring programs, including the Puget

Sound Estuary Program (1990c, 1990d), the NOAA Mussel Watch Program

(Battelle, 1989), and the California Mussel Watch Program (California Department

of Fish and Game, 1990), recommend different procedures for processing

samples for organics and metals analyses. However, this may not be feasible if

fish are too small to allow for preparing separate composites from individual fillets

or if resources are limited. If a single composite sample is prepared for the

analyses of both organics and metals, precautions must be taken to use materials

and cleaning procedures that are noncontaminating for both organics and metals.

Quartz, ceramic, borosilicate glass, and PTFE are recommended materials for

sample processing equipment. If chromium and nickel are not of concern, highquality, corrosion-resistant stainless steel utensils may be used. Knives with

titanium blades and PTFE handles are recommended for performing tissue

resections (Lowenstein and Young, 1986). Borosilicate glass bench liners are

recommended. Filleting should be done on glass or PTFE cutting boards that are

cleaned properly between fish or on cutting boards covered with heavy duty

aluminum foil that is changed after each filleting. Fillets or tissue homogenates

should be stored in clean borosilicate glass, quartz, or PTFE containers with

PTFE-lined lids.

Prior to preparing each composite sample, utensils and containers should be

cleaned thoroughly with a detergent solution, rinsed with tap water, soaked in

50 percent HNO3, for 12 to 24 hours at room temperature, and then rinsed with

organics- and metal-free water. Note: Chromic acid should not be used for

cleaning any materials. Acids used should be at least reagent grade. Stainless

steel parts may be cleaned using this recommended procedure with the acid

soaking step method omitted (Stober, 1991).

Aliquots of composite homogenates taken for metals analysis (see Section 7.3.1)

may be stored in plastic containers that have been cleaned according to the
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-7

procedure outlined above, with the exception that aqua regia must not be used for

the acid soaking step.

7.2.2 Processing Fish Samples

Processing in the laboratory to prepare fish fillet composite homogenate samples

for analysis (diagrammed in Figure 7-1) involves

• Inspecting individual fish

• Weighing individual fish

• Removing scales and/or otoliths for age determination (optional)

• Determining the sex of each fish (optional)

• Examining each fish for morphological abnormalities (optional)

• Scaling all fish with scales (leaving belly flap on); removing skin of scaleless

fish (e.g., catfish)

• Filleting (resection)

• Weighing fillets

• Homogenizing fillets

• Preparing a composite homogenate

• Preparing aliquots of the composite homogenate for analysis

• Distributing frozen aliquots to one or more analytical laboratories.

Whole fish should be shipped or brought to the sample processing laboratory from

the field on wet or blue ice within 24 hours of sample collection. Fillets should be

resected within 48 hours of sample collection. Ideally, fish should not be frozen

prior to resection because freezing may cause internal organs to rupture and

contaminate edible tissue (Stober, 1991; U.S. EPA, 1986b). However, if resection

cannot be performed within 48 hours, the whole fish should be frozen at the

sampling site and shipped to the sample processing laboratory on dry ice. Fish

samples that arrive frozen (i.e., on dry ice) at the sample processing laboratory

should be placed in a 20 C freezer for storage until filleting can be performed.

The fish should then be partially thawed prior to resection. Note: If the fillet tissue

is contaminated by materials released from the rupture of the internal organs

during freezing, the state may eliminate the fillet tissue as a sample or, alternatively, the fillet tissues should be rinsed in contaminant-free, distilled deionized
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-8

Composite equal weights (g) of 

homogenized fillet tissues from the 

selected number of fish (200-g)

Seal and label (200-g) composite 

homogenate in appropriate container(s) 

and store at ≤-20 °C until analysis (see 

Table 7-1 for recommended container 

materials and holding times).

Log in fish samples using COC procedures

Unwrap and inspect individual fish

Weigh individual fish

Remove and archive scales and/or otoliths for age determination (optional)

Determine sex (optional); note morphological abnormalities (optional)

Save remainder of fillet

homogenate from each fish

Seal and label individual fillet 

homogenates in appropriate 

container(s) and archive at 

≤-20 °C (see Table 7-1 for 

recommended container 

materials and holding times).

Remove scales from all scaled fish Remove skin from scaleless fish (e.g., catfish) 

COC = Chain of custody.

Fillet fish

Weigh fillets (g)

Homogenize fillets

Divide homogenized sample into quarters, mix opposite

quarters, and then mix halves (3 times)

Optional

Figure 7-1. Preparation of fish fillet composite homogenate samples.
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-9

water and blotted dry. Regardless of the procedure selected, a notation should

be made in the sample processing record.

Sample processing procedures are discussed in the following sections. Data from

each procedure should be recorded directly in a bound laboratory notebook or on

forms that can be secured in the laboratory notebook. A sample processing

record for fish fillet composites is shown in Figure 7-2.

7.2.2.1 Sample Inspection—

Individual fish received for filleting should be unwrapped and inspected carefully

to ensure that they have not been compromised in any way (i.e., not properly

preserved during shipment). Any specimen deemed unsuitable for further

processing and analysis should be discarded and identified on the sample

processing record.

7.2.2.2 Sample Weighing—

A wet weight should be determined for each fish. All samples should be weighed

on balances that are properly calibrated and of adequate accuracy and precision

to meet program data quality objectives. Balance calibration should be checked

at the beginning and end of each weighing session and after every 20 weighings

in a weighing session. 

Fish shipped on wet or blue ice should be weighed directly on a foil-lined balance

tray. To prevent cross contamination between individual fish, the foil lining should

be replaced after each weighing. Frozen fish (i.e., those shipped on dry ice)

should be weighed in clean, tared, noncontaminating containers if they will thaw

before the weighing can be completed. Note: Liquid from the thawed whole fish

sample will come not only from the fillet tissue but from the gut and body cavity,

which are not part of the final fillet sample. Consequently, inclusion of this liquid

with the sample may result in an overestimate of target analyte and lipid

concentrations in the fillet homogenate. Nevertheless, it is recommended, as a

conservative approach, that all liquid from the thawed whole fish sample be kept

in the container as part of the sample.

All weights should be recorded to the nearest gram on the sample processing

record and/or in the laboratory notebook.

7.2.2.3 Age Determination (Optional)—

Age provides a good indication of the duration of exposure to pollutants (Versar,

1982). A few scales or otoliths (Jearld, 1983) should be removed from each fish

and delivered to a fisheries biologist for age determination. For most warm water

inland gamefish, 5 to 10 scales should be removed from below the lateral line and

behind the pectoral fin. On soft-rayed fish such as trout and salmon, the scales

should be taken just above the lateral line (WDNR, 1988). For catfish and other
7-10Figure 7-2. Sample processing record for fish contaminant monitoring program

—fish fillet comp
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-11

scaleless fish, the pectoral fin spines should be clipped and saved (Versar, 1982).

The scales, spines, or otoliths may be stored by sealing them in small envelopes

(such as coin envelopes) or plastic bags labeled with, and cross-referenced by,

the identification number assigned to the tissue specimen (Versar, 1982).

Removal of scales, spines, or otoliths from each fish should be noted (by a check

mark) on the sample processing record.

7.2.2.4 Sex Determination (Optional)—

Fish sex should be determined before filleting. To determine the sex of a fish, an

incision should be made on the ventral surface of the body from a point

immediately anterior to the anus toward the head to a point immediately posterior

to the pelvic fins. If necessary, a second incision should be made on the left side

of the fish from the initial point of the first incision toward the dorsal fin. The

resulting flap should be folded back to observe the gonads. Ovaries appear

whitish to greenish to golden brown and have a granular texture. Testes appear

creamy white and have a smooth texture (Texas Water Commission, 1990). The

sex of each fish should be recorded on the sample processing form.

7.2.2.5 Assessment of Morphological Abnormalities (Optional)—

Assessment of gross morphological abnormalities in finfish is optional. This

assessment may be conducted in the field (see Section 6.3.1.5) or during initial

inspection at the processing laboratory prior to filleting. States interested in

documenting morphological abnormalities should consult Sinderman (1983) and

review recommended protocols for fish pathology studies used in the Puget

Sound Estuary Program (1990c) and those described by Goede and Barton

(1990).

7.2.2.6 Scaling or Skinning—

To control contamination, separate sets of utensils and cutting boards should be

used for skinning or scaling fish and for filleting fish. Fish with scales should be

scaled and any adhering slime removed prior to filleting. Fish without scales (e.g.,

catfish) should be skinned prior to filleting. These fillet types are recommended

because it is believed that they are most representative of the edible portions of

fish prepared and consumed by sport anglers. However, it is the responsibility of

each program manager, in consultation with state fisheries experts, to select the

fillet or sample type most appropriate for each target species based on the dietary

customs of local populations of concern. 

A fish is scaled by laying it flat on a clean glass or PTFE cutting board or on one

that has been covered with heavy duty aluminum foil and removing the scales and

adhering slime by scraping from the tail to the head using the blade edge of a

clean stainless steel, ceramic, or titanium knife. Cross-contamination is controlled

by rinsing the cutting board and knife with contaminant-free distilled water

between fish. If an aluminum-foil-covered cutting board is used, the foil should be
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-12

changed between fish. The skin should be removed from fish without scales by

loosening the skin just behind the gills and pulling it off between knife blade and

thumb or with pliers as shown in Figure 7-3.

Once the scales and slime have been scraped off or the skin removed, the

outside of the fish should be washed with contaminant-free distilled water and it

should be placed on a second clean cutting board for filleting.

7.2.2.7 Filleting—

Filleting should be conducted only by or under the supervision of an experienced

fisheries biologist. If gloves are worn, they should be talc- or dust-free, and of

noncontaminating materials. Prior to filleting, hands should be washed with Ivory

soap and rinsed thoroughly in tap water, followed by distilled water (U.S. EPA,

1991d). Specimens should come into contact with noncontaminating surfaces

only. Fish should be filleted on glass or PTFE cutting boards that are cleaned

properly between fish or on cutting boards covered with heavy duty aluminum foil

that is changed between fish (Puget Sound Estuary Program, 1990d, 1990e).

Care must be taken to avoid contaminating fillet tissues with material released

from inadvertent puncture of internal organs. Note: If the fillet tissue is

contaminated by materials released from the inadvertent puncture of the internal

organs during resection, the state may eliminate the fillet tissue as a sample or,

alternatively, the fillet tissue should be rinsed in contaminant-free, deionized

distilled water and blotted dry. Regardless of the procedure selected, a notation

should be made in the sample processing record.

Ideally, fish should be filleted while ice crystals are still present in the muscle

tissue. Therefore, if fish have been frozen, they should not be allowed to thaw

completely prior to filleting. Fish should be thawed only to the point where it

becomes possible to make an incision into the flesh (U.S. EPA, 1991d). 

Clean, high-quality stainless steel, ceramic, or titanium utensils should be used

to remove one or both fillets from each fish, as necessary. The general procedure

recommended for filleting fish is illustrated in Figure 7-3 (U.S. EPA, 1991d). 

The belly flap should be included in each fillet. Any dark muscle tissue in the

vicinity of the lateral line should not be separated from the light muscle tissue that

constitutes the rest of the muscle tissue mass. Bones still present in the tissue

after filleting should be removed carefully (U.S. EPA, 1991d).

If both fillets are removed from a fish, they can be combined or kept separate for

duplicate QC analysis, analysis of different analytes, or archival of one fillet.

Fillets should be weighed (either individually or combined, depending on the

analytical requirements) and the weight(s) recorded to the nearest gram on the

sample processing record. 
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-13

Source: U.S. EPA, 1991d.

Figure 7-3. Illustration of basic fish filleting procedure.
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-14

If fillets are to be homogenized immediately, they should be placed in a properly

cleaned glass or PTFE homogenization container. If samples are to be analyzed

for metals only, plastic homogenization containers may be used. To facilitate

homogenization, it may be necessary or desirable to chop each fillet into smaller

pieces using a titanium or stainless steel knife prior to placement in the

homogenization container.

If fillets are to be homogenized later, they should be wrapped in heavy duty

aluminum foil and labeled with the sample identification number, the sample type

(e.g., "F" for fillet), the weight (g), and the date of resection. If composite

homogenates are to be prepared from only a single fillet from each fish, fillets

should be wrapped separately and the designation "F1" and "F2" should be added

to the sample identification number for each fillet. The individual fillets from each

fish should be kept together. All fillets from a composite sample should be placed

in a plastic bag labeled with the composite identification number, the individual

sample identification numbers, and the date of resection and stored at -20 C

until homogenization.

7.2.2.8 Preparation of Individual Homogenates—

To ensure even distribution of contaminants throughout tissue samples and to

facilitate extraction and digestion of samples, the fillets from individual fish must

be ground and homogenized prior to analysis. The fillets from an individual fish

may be ground and homogenized separately or combined, depending on the

analytical requirements and the sample size.

Fish fillets should be ground and homogenized using an automatic grinder or highspeed blender or homogenizer. Large fillets may be cut into 2.5-cm cubes with

high-quality stainless steel or titanium knives or with a food service band saw prior

to homogenization. Parts of the blender or homogenizer used to grind the tissue

(i.e., blades, probes) should be made of tantalum or titanium rather than stainless

steel. Stainless steel blades and/or probes have been found to be a potential

source of nickel and chromium contamination (due to abrasion at high speeds)

and should be avoided.

Grinding and homogenization of tissue is easier when it is partially frozen (Stober,

1991). Chilling the grinder/blender briefly with a few chips of dry ice will also help

keep the tissue from sticking to it (Smith, 1985).

The fillet sample should be ground until it appears to be homogeneous. The

ground sample should then be divided into quarters, opposite quarters mixed

together by hand, and the two halves mixed together. The grinding, quartering,

and hand-mixing steps should be repeated at least two more times. If chunks of

tissue are present at this point, the grinding and homogenization should be

repeated. Note: Skin-on fillets are the fish fillet sample type recommended for

use in state fish contaminant monitoring programs. However, skin-on fillets of

some finfish species are especially difficult to homogenize completely. No chunks
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-15

of tissue or skin should remain in the sample homogenate because these may not

be extracted or digested efficiently and could bias the analytical results. If

complete homogenization of skin-on fillets for a particular target species is a

chronic problem or if local consumers are likely to prepare skinless fillets of the

species, the state should consider analyzing skinless fillet samples. If the sample

is to be analyzed for metals only, the ground tissue may be mixed by hand in a

polyethylene bag (Stober, 1991). The preparation of each individual homogenate

should be noted (marked with a check) on the sample processing record. At this

time, individual homogenates may be either processed further to prepare

composite homogenates or frozen separately and stored at -20 C (see

Table 7-1).

7.2.2.9 Preparation of Composite Homogenates—

Composite homogenates should be prepared from equal weights of individual

homogenates. The same type of individual homogenate (i.e., either single fillet

or combined fillet) should always be used in a given composite sample.

If individual homogenates have been frozen, they should be thawed partially and

rehomogenized prior to weighing and compositing. Any associated liquid should

be kept as a part of the sample. The weight of each individual homogenate used

in the composite homogenate should be recorded, to the nearest gram, on the

sample processing record.

Each composite homogenate should be blended as described for individual

homogenates in Section 7.2.2.8. The composite homogenate may be processed

immediately for analysis or frozen and stored at -20 C (see Table 7-1).

The remainder of each individual homogenate should be archived at -20 C with

the designation "Archive" and the expiration date recorded on the sample label.

The location of the archived samples should be indicated on the sample

processing record under "Notes."

It is essential that the weights of individual homogenates yield a composite

homogenate of adequate size to perform all necessary analyses. Weights of

individual homogenates required for a composite homogenate, based on the

number of fish per composite and the weight of composite homogenate

recommended for analyses of all screening study target analytes (see Table 4-1),

are given in Table 7-2. The total composite weight required for intensive studies

may be less than that for screening studies if the number of target analytes is

reduced significantly.

The recommended sample size of 200 g for screening studies is intended to

provide sufficient sample material to (1) analyze for all recommended target

analytes (see Table 4-1) at appropriate detection limits; (2) meet minimum QC

requirements for the analyses of laboratory duplicate, matrix spike, and matrix

spike duplicate samples (see Sections 8.3.3.4 and 8.3.3.5); and (3) allow for
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-16

Table 7-2. Weights (g) of Individual Homogenates

Required for Screening Study Composite Homogenate Samplea,b

Number of

fish per sample

Total composite weight

100 g

(minimum)

200 g

(recommended)

500 g

(maximum)

3 33 67 167

4 25 50 125

5 20 40 100

6 17 33 84

7 14 29 72

8 13 25 63

9 11 22 56

10 10 20 50

a Based on total number of fish per composite and the total composite weight required for

analysis in screening studies. The total composite weight required in intensive studies may be

less if the number of target analytes is reduced significantly. b Individual homogenates may be prepared from one or both fillets from a fish. A composite

homogenate should be prepared only from individual homogenates of the same type (i.e.,

either from individual homogenates each prepared from a single fillet or from individual

homogenates each prepared from both fillets).

reanalysis if the QC control limits are not met or if the sample is lost. However,

sample size requirements may vary among laboratories and the analytical

methods used. Each program manager must consult with the analytical

laboratory supervisor to determine the actual weights of composite homogenates

required to analyze for all selected target analytes at appropriate detection limits.

7.2.3 Processing Turtle Samples

Processing in the laboratory to prepare individual turtle homogenate samples for

analysis (diagrammed in Figure 7-4) involves 

• Inspecting individual turtles

• Weighing individual turtles

• Removing edible tissues

• Determining the sex of each turtle (optional)

• Determining the age of each turtle (optional)

• Weighing edible tissue or tissues

• Homogenizing tissues

• Preparing individual homogenate samples

• Preparing aliquots of the individual homogenates for analysis

• Distributing frozen aliquots to one or more analytical laboratories.
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-17

Weigh edible tissue (g)

(muscle with or without other internal tissues added)

Homogenize edible tissue sample

Divide homogenized sample into quarters, mix opposite

quarters, and then mix halves (3 times)

Seal and label (200-g) 

individual homogenate in 

appropriate container(s) 

and store at ≤-20 °C until 

analysis (see Table 7-1 for 

recommended container 

materials and holding 

times).

Log in turtle samples using COC procedures

Remove turtle from bag and inspect turtle

Weigh individual turtle

Sever bony bridges on ventral side; remove plastron

Weigh heart, liver, fatty deposits, and eggs 

separately (g)

Homogenize individual tissue types separately

Divide homogenized sample of each tissue type 

into quarters, mix opposite quarters, and then 

mix halves (3 times)

Seal and label individual tissue homogenates in 

appropriate container(s) and archive at ≤-20 °C 

until analysis (see Table 7-1 for recommended 

container materials and holding times).

Resect forelimbs, hindlimbs, neck, and tail muscle tissue from the body. 

Skin all muscle tissue, remove claws and bones. Also resect muscle 

tissue inside carapace. NOTE: Depending on dietary practices of 

population of concern, add heart, liver, fatty tissues, and eggs to 

muscle sample or, alternatively, retain these other tissues for separate 

analysis.

COC = Chain of custody.

Seal and label remaining 

individual homogenate in 

appropriate container(s) 

and store at ≤-20 °C until 

analysis (see Table 7-1 for 

recommended container 

materials and holding 

times).

Optional

Determine the sex of each turtle (optional)

Retain bones for age determination (optional)

Figure 7-4. Preparation of individual turtle homogenate samples.
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-18

Whole turtles should be shipped or brought to the sample processing laboratory

from the field on wet or blue ice within 24 hours of sample collection. The

recommended euthanizing method for turtles is freezing (Frye, 1994) and a

minimum of 48 hours or more may be required for large specimens. Turtles that

arrive on wet or blue ice or frozen (i.e., on dry ice) at the sample processing

laboratory should be placed in a -20 C freezer for storage until resection can

be performed. If rupture of internal organs is noted for an individual turtle, the

specimen may be eliminated as a sample or, alternatively, the edible tissues

should be rinsed in distilled deionized water and blotted dry.

Sample processing procedures are discussed in the following sections. Data from

each procedure should be recorded directly in a bound laboratory notebook or on

forms that can be secured in the laboratory notebook. A sample processing

record for individual turtle samples is shown in Figure 7-5.

7.2.3.1 Sample Inspection—

Turtles received for resection should be removed from the canvas or burlap

collection bags and inspected carefully to ensure that they have not been

compromised in any way (i.e., not properly preserved during shipment). Any

specimen deemed unsuitable for further processing and analysis should be

discarded and identified on the sample processing record.

7.2.3.2 Sample Weighing—

A wet weight should be determined for each turtle. All samples should be

weighed on balances that are properly calibrated and of adequate accuracy and

precision to meet program data quality objectives. Balance calibration should be

checked at the beginning and end of each weighing session and after every 20

weighings in a weighing session. 

Turtles euthanized by freezing should be weighed in clean, tared, noncontaminating containers if they will thaw before the weighing can be completed.

Note: Liquid from the thawed whole turtle sample will come not only from the

muscle tissue but from the gut and body cavity, which may not be part of the

desired edible tissue sample. Consequently, inclusion of this liquid with the

sample may result in an overestimate of target analyte and lipid concentrations

in the edible tissue homogenate. Nevertheless, it is recommended, as a

conservative approach, that all liquid from the thawed whole turtle be kept in the

container as part of the sample.

All weights should be recorded to the nearest gram on the sample processing

record and/or in the laboratory notebook.
7-19Figure 7-5. Sample processing record for a contaminant monitoring program

—individual turtle s
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-20

7.2.3.3 Removal of Edible Tissues—

Edible portions of a turtle should consist only of those tissues that the population

of concern might reasonably be expected to eat. Edible tissues should be clearly

defined in site-specific sample processing protocols. A brief description of the

edible portions used should also be provided on the sample processing record.

General procedures for removing edible tissues from a turtle are illustrated in

Appendix K.

Resection should be conducted only by or under the supervision of an

experienced fisheries biologist. If gloves are worn, they should be talc- or dustfree and of noncontaminating materials. Prior to resection, hands should be

washed with soap and rinsed thoroughly in tap water, followed by distilled water

(U.S. EPA, 1991d). Specimens should come into contact with noncontaminating

surfaces only. Turtles should be resected on glass or PTFE cutting boards that

are cleaned properly between each turtle or on cutting boards covered with heavy

duty aluminum foil that is changed between each turtle (Puget Sound Estuary

Program, 1990d, 1990e). A turtle is resected by laying it flat on its back and

removing the plastron by severing the two bony ridges between the forelimbs and

hindlimbs. Care must be taken to avoid contaminating edible tissues with material

released from the inadvertent puncture of internal organs.

Ideally, turtles should be resected while ice crystals are still present in the muscle

tissue. Thawing of frozen turtles should be kept to a minimum during tissue

removal to avoid loss of liquids. A turtle should be thawed only to the point where

it becomes possible to make an incision into the flesh (U.S. EPA, 1991d).

Clean, high-quality stainless steel, ceramic, or titanium utensils should be used

to remove the muscle tissue and, depending on dietary or culinary practices of the

population of concern, some of the other edible tissues from each turtle. The

general procedure recommended for resecting turtles is illustrated in Figure 7-6.

Skin on the forelimbs, hindlimbs, neck, and tail should be removed. Claws should

be removed from the forelimbs and hindlimbs. Bones still present in the muscle

tissue after resection should be removed carefully (U.S. EPA, 1991d) and may be

used in age determination (see Section 7.2.3.5).

To control contamination, separate sets of utensils and cutting boards should be

used for skinning muscle tissue and resecting other internal tissues from the turtle

(e.g., heart, liver, fatty deposits, and eggs). These other tissue types are

recommended for inclusion with the muscle tissue as part of the edible tissue

sample because it is believed that they are most representative of the edible

portions of turtles that are prepared and consumed by sport anglers and

subsistence fishers. Alternatively, states may choose to analyze some of these

other lipophilic tissues separately. It is the responsibility of each program

manager, in consultation with state fisheries experts, to select the tissue sample
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-21

Figure 7-6. Illustration of basic turtle resection procedure.

type most appropriate for each target species based on the dietary customs of

local populations of concern. 

The edible turtle tissues should be weighed and the weight recorded to the

nearest gram on the sample processing record. If the state elects to analyze the

heart, liver, fatty deposits, or eggs separately from the muscle tissue, these other

tissues should be weighed separately and the weights recorded to the nearest

gram in the sample processing record.

If the tissues are to be homogenized immediately, they should be placed in a

properly cleaned glass or PTFE homogenization container. If samples are to be

analyzed for metals only, plastic homogenization containers may be used. To

facilitate homogenization, it may be necessary or desirable to chop each of the

large pieces of muscle tissue into smaller pieces using a titanium or stainless steel

knife prior to placement in the homogenization container.

If the tissues are to be homogenized later, they should be wrapped in heavy duty

aluminum foil and labeled with the sample identification number, the sample type

(e.g., "M" for muscle, "E" for eggs, or "FD" for fatty deposits), the weight (g), and

the date of resection. The individual muscle tissue samples from each turtle

should be packaged together and given an individual sample identification

number. The date of resection should be recorded and the sample should be

stored at -20C until homogenization. Note: State staff may determine that the

most appropriate sample type is muscle tissue only, with internal organ tissues

analyzed separately (liver, heart, fatty deposits, or eggs). Alternatively, state staff

may determine that the most appropriate sample type is muscle tissue with

several other internal organs included as the turtle tissue sample. This latter
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-22

sample type typically will provide a more conservative estimate of contaminant

residues, particularly with respect to lipophilic target analytes (e.g., PCBs, dioxins,

and organochlorine pesticides).

7.2.3.4 Sex Determination (Optional)—

Turtle sex should be determined during resection if it has not already been

determined in the field. Once the plastron is removed, the ovaries or testes can

be observed posterior and dorsal to the liver. Each ovary is a large egg-filled sac

containing yellow spherical eggs in various stages of development (Ashley, 1962)

(see Appendix K). Each testes is a spherical organ, yellowish in color, attached

to the ventral side of each kidney. The sex of each turtle should be verified and

recorded on the sample processing form.

7.2.3.5 Age Determination (Optional)—

Age provides a good indication of the duration of exposure to pollutants (Versar,

1982). Several methods have been developed for estimating the age of turtles

(Castanet, 1994; Frazer et al., 1993; Gibbons, 1976). Two methods are

appropriate for use in contaminant monitoring programs where small numbers of

animals of a particular species are to be collected and where the animals must be

sacrificed for tissue residue analysis. These methods include (1) the use of

external annuli (scute growth marks) on the plastron and (2) the use of growth

rings on the bones.

The surface of epidermal keratinous scutes on the plastron of turtle shells

develops successive persistent grooves or growth lines during periods of slow or

arrested growth (Zangerl, 1969). Because these growth rings are fairly obvious,

they have been used extensively for estimating age in various turtle species

(Cagle, 1946, 1948, 1950; Gibbons, 1968; Legler, 1960; Sexton, 1959). This

technique is particularly useful for younger turtles where the major growth rings

are more definitive and clear cut than in older individuals (Gibbons, 1976).

However, a useful extension of the external annuli method is presented by Sexton

(1959) showing that age estimates can be made for adults on which all annuli are

not visible. This method involves visually examining the plastron of the turtle

during the resection or tagging the plastron with the sample identification number

of the turtle and retaining it for later analysis.

The use of bone rings is the second method that may be used to estimate age in

turtles (Enlow and Brown, 1969; Peabody, 1961). Unlike the previous visual

method, this method requires that the bones of the turtle be removed during

resection and retained for later analysis. The growth rings appear at the surface

or inside primary compacta of bone tissues. There are two primary methods for

observing growth marks: either directly at the surface of the bone as in flat bones

using transmitted or reflected light or inside the long bones using thin sections

(Castanet, 1994; Dobie, 1971; Galbraith and Brooks, 1987; Hammer, 1969;

Gibbons, 1976; Mattox, 1935; Peabody, 1961). The methods of preparation of
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-23

whole bones and histological sections of fresh material for growth mark

determinations are now routinely performed. Details of these methods can be

found in Castanet (1974 and 1987), Castanet et al. (1993), and Zug et al. (1986).

State staff interested in using either of these methods for age determination of

turtles should read the review articles by Castanet (1994) and Gibbons (1976) for

discussions of the advantages and disadvantages of each method, and the

associated literature cited in these articles on turtle species of particular interest

within their jurisdictions.

7.2.3.6 Preparation of Individual Homogenates—

To ensure even distribution of contaminants throughout tissue samples and to

facilitate extraction and digestion of samples, the edible tissues from individual

turtles must be ground and homogenized prior to analysis. The various tissues

from an individual turtle may be ground and homogenized separately, or

combined, depending on the sampling program’s definition of edible tissues.

Turtle tissues should be ground and homogenized using an automatic grinder or

high-speed blender or homogenizer. Large pieces of muscle or organ tissue (e.g.,

liver or fatty deposits) may be cut into 2.5-cm cubes with high-quality stainless

steel or titanium knives or with a food service band saw prior to homogenization.

Parts of the blender or homogenizer used to grind the tissue (i.e., blades, probes)

should be made of tantalum or titanium rather than stainless steel. Stainless steel

blades and/or probes have been found to be a potential source of nickel and

chromium contamination (due to abrasion at high speeds) and should be avoided.

Grinding and homogenization of tissue is easier when it is partially frozen (Stober,

1991). Chilling the grinder/blender briefly with a few chips of dry ice will also help

keep the tissue from sticking to it (Smith, 1985).

The tissue sample should be ground until it appears to be homogeneous. The

ground sample should then be divided into quarters, opposite quarters mixed

together by hand, and the two halves mixed together. The grinding, quartering,

and hand-mixing steps should be repeated at least two more times. If chunks of

tissue are present at this point, the grinding and homogenization should be

repeated. No chunks of tissue should remain because these may not be

extracted or digested efficiently and could bias the analytical results. This is

particularly true when lipophilic tissues (e.g., fatty deposits, liver, or eggs) are not

completely homogenized throughout the sample. Portions of the tissue sample

that retain unhomogenized portions of tissues may exhibit higher or lower

residues of target analytes than properly homogenized samples.

If the sample is to be analyzed for metals only, the ground tissue may be mixed

by hand in a polyethylene bag (Stober, 1991). The preparation of each individual

homogenate should be noted (marked with a check) on the sample processing

record. At this time, individual homogenates may be frozen separately and stored

at -20 C (see Table 7-1).
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-24

The remainder of each individual homogenate should be archived at -20 C with

the designation "Archive" and the expiration date recorded on the sample label.

The location of the archived samples should be indicated on the sample

processing record under "Notes."

It is essential that the weight of individual homogenate samples is of adequate

size to perform all necessary analyses. The recommended sample size of 200

g for screening studies is intended to provide sufficient sample material to (1)

analyze for all recommended target analytes (see Table 4-1) at appropriate

detection limits; (2) meet minimum QC requirements for the analyses of laboratory

duplicate, matrix spike, and matrix spike duplicate samples (see Sections 8.3.3.4

and 8.3.3.5); and (3) allow for reanalysis if the QC control limits are not met or if

the sample is lost. However, sample size requirements may vary among

laboratories and the analytical methods used. Each program manager must

consult with the analytical laboratory supervisor to determine the actual weights

of homogenates required to analyze for all selected target analytes at appropriate

detection limits. The total sample weight required for intensive studies may be

less than that for screening studies if the number of target analytes is reduced

significantly.

7.2.4 Processing Shellfish Samples

Laboratory processing of shellfish to prepare edible tissue composite

homogenates for analysis (diagrammed in Figure 7-7) involves 

• Inspecting individual shellfish

• Determining the sex of each shellfish (optional)

• Examining each shellfish for morphological abnormalities (optional)

• Removing the edible parts from each shellfish in the composite sample (3 to

50 individuals, depending upon the species)

• Combining the edible parts in an appropriate noncontaminating container

• Weighing the composite sample

• Homogenizing the composite sample

• Preparing aliquots of the composite homogenate for analysis

• Distributing frozen aliquots to one or more analytical laboratories.

Sample aliquotting and shipping are discussed in Section 7.3; all other processing

steps are discussed in this section. Shellfish samples should be processed

following the general guidelines in Section 7.2.1 to avoid contamination. In
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-25

Log in shellfish samples using COC procedures

Unwrap and inspect individual shellfish

Determine sex (optional); note morphological 

abnormalities (optional)

Remove edible tissue from each shellfish in composite

Combine edible tissue from individual shellfish in 

composite in a tared container (g)

Weigh the filled container (g)

Homogenize the composite sample

Divide homogenized sample into quarters, mix opposite 

quarters and then mix halves (3 times)

Seal and label remaining 

composite homogenate in 

appropriate container(s) and 

archive at ≤-20 °C (see Table 7-1 

for recommended container 

materials and holding times).

Seal and label (200-g) composite 

homogenate in appropriate 

container(s) and store at ≤-20 °C 

until analysis (see Table 7-1 for 

recommended container materials 

and holding times).

COC = Chain of custody.

Figure 7-7. Preparation of shellfish edible tissue composite homogenate samples.
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-26

particular, it is recommended that separate composite homogenates be prepared

for the analysis of metals and organics if resources allow. A sample processing

record for shellfish edible tissue composite samples is shown in Figure 7-8.

Shellfish samples should be shipped or brought to the sample processing

laboratory either on wet or blue ice (if next-day delivery is assured) or on dry ice

(see Section 6.3.3). Shellfish samples arriving on wet ice or blue ice should have

edible tissue removed and should be frozen to -20C within 48 hours after

collection. Shellfish samples that arrive frozen (i.e., on dry ice) at the processing

laboratory should be placed in a -20C freezer for storage until edible tissue is

removed.

7.2.4.1 Sample Inspection—

Individual shellfish should be unwrapped and inspected carefully to ensure that

they have not been compromised in any way (i.e., not properly preserved during

shipment). Any specimen deemed unsuitable for further processing and analysis

should be discarded and identified on the sample processing record.

7.2.4.2 Sex Determination (Optional)—

The determination of sex in shellfish species is impractical if large numbers of

individuals of the target species are required for each composite sample.

For bivalves, determination of sex is a time-consuming procedure that must be

performed after shucking but prior to removal of the edible tissues. Once the

bivalve is shucked, a small amount of gonadal material can be removed using a

Pasteur pipette. The gonadal tissue must then be examined under a microscope

to identify egg or sperm cells.

For crustaceans, sex also should be determined before removal of the edible

tissues. For many species, sex determination can be accomplished by visual

inspection. Sexual dimorphism is particularly striking in many species of

decapods. In the blue crab, Callinectes sapidus, the female has a broad

abdomen suited for retaining the maturing egg mass or sponge, while the

abdomen of the male is greatly reduced in width. For shrimp, lobsters, and crayfish, sexual variations in the structure of one or more pair of pleopods are

common. States interested in determining the sex of shellfish should consult

taxonomic keys for specific information on each target species.

7.2.4.3 Assessment of Morphological Abnormalities (Optional)—

Assessment of gross morphological abnormalities in shellfish is optional. This

assessment may be conducted in the field (see Section 6.3.1.5) or during initial

inspection at the processing laboratory prior to removal of the edible tissues.

States interested in documenting morphological abnormalities should consult

Sinderman and Rosenfield (1967), Rosen (1970), and Murchelano (1982) for
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-27

Figure 7-8. Sample processing record for shellfish contaminant monitoring

program—edible tissue composites.
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-28

detailed information on various pathological conditions in shellfish and review

recommended protocols for pathology studies used in the Puget Sound Estuary

Program (1990c).

7.2.4.4 Removal of Edible Tissue—

Edible portions of shellfish should consist only of those tissues that the population

of concern might reasonably be expected to eat. Edible tissues should be clearly

defined in site-specific sample processing protocols. A brief description of the

edible portions used should also be provided on the sample processing record.

General procedures for removing edible tissues from a variety of shellfish are

illustrated in Appendix L.

Thawing of frozen shellfish samples should be kept to a minimum during tissue

removal to avoid loss of liquids. Shellfish should be rinsed well with organics- and

metal-free water prior to tissue removal to remove any loose external debris.

Bivalve molluscs (oysters, clams, mussels, and scallops) typically are prepared

by severing the adductor muscle, prying open the shell, and removing the soft

tissue. The soft tissue includes viscera, meat, and body fluids (Smith, 1985).

Byssal threads from mussels should be removed with a knife before shucking and

should not be included in the composite sample. 

Edible tissue for crabs typically includes all leg and claw meat, back shell meat,

and body cavity meat. Internal organs generally are removed. Inclusion of the

hepatopancreas should be determined by the eating habits of the local population

or subpopulations of concern. If the crab is soft-shelled, the entire crab should be

used in the sample. Hard- and soft-shelled crabs must not be combined in the

same composite (Smith, 1985). 

Typically, shrimp and crayfish are prepared by removing the cephalothorax and

then removing the tail meat from the shell. Only the tail meat with the section of

intestine passing through the tail muscle is retained for analysis (Smith, 1985).

Edible tissue for lobsters typically includes the tail and claw meat. If the tomalley

(hepatopancreas) and gonads or ovaries are consumed by local populations of

concern, these parts should also be removed and analyzed separately (Duston

et al., 1990).

7.2.4.5 Sample Weighing—

Edible tissue from all shellfish in a composite sample (3 to 50 individuals) should

be placed in an appropriate preweighed and labeled noncontaminating container.

The weight of the empty container (tare weight) should be recorded to the nearest

gram on the sample processing record. All fluids accumulated during removal of

edible tissue should be retained as part of the sample. As the edible portion of

each shellfish is placed in the container, it should be noted on the sample

processing record. When the edible tissue has been removed from all shellfish
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-29

in the composite, the container should be reweighed and the weight recorded to

the nearest gram on the sample processing record. The total composite weight

should be approximately 200 g for screening studies. If the number of target

analytes is significantly reduced in intensive studies, a smaller composite

homogenate sample may suffice (see Section 7.2.2.9). At this point, the

composite sample may be processed for analysis or frozen and stored at -20C

(see Table 7-1).

7.2.4.6 Preparation of Composite Homogenates—

Composite samples of the edible portions of shellfish should be homogenized in

a grinder, blender, or homogenizer that has been cooled briefly with dry ice

(Smith, 1985). For metals analysis, tissue may be homogenized in 4-oz

polyethylene jars (California Department of Fish and Game, 1990) using a

Polytron equipped with a titanium generator. If the tissue is to be analyzed for

organics only, or if chromium and nickel contamination are not of concern, a

commercial food processor with stainless steel blades and glass container may

be used. The composite should be homogenized to a paste-like consistency.

Larger samples may be cut into 2.5-cm cubes with high-quality stainless steel or

titanium knives before grinding. If samples were frozen after dissection, they can

be cut without thawing with either a knife-and-mallet or a clean bandsaw. The

ground samples should be divided into quarters, opposite quarters mixed together

by hand, and the two halves mixed together. The quartering and mixing should

be repeated at least two more times until a homogeneous sample is obtained. No

chunks should remain in the sample because these may not be extracted or

digested efficiently. At this point, the composite homogenates may be processed

for analysis or frozen and stored at -20 C (see Table 7-1).

7.3 SAMPLE DISTRIBUTION

The sample processing laboratory should prepare aliquots of the composite

homogenates for analysis, distribute the aliquots to the appropriate laboratory (or

laboratories), and archive the remainder of each composite homogenate.

7.3.1 Preparing Sample Aliquots

Note: Because lipid material tends to migrate during freezing, frozen composite

homogenates must be thawed and rehomogenized before aliquots are prepared

(U.S. EPA, 1991d). Samples may be thawed overnight in an insulated cooler or

refrigerator and then homogenized. Recommended aliquot weights and

appropriate containers for different types of analyses are shown in Table 7-3. The

actual sample size required will depend on the analytical method used and the

laboratory performing the analysis. Therefore, the exact sample size required for

each type of analysis should be determined in consultation with the analytical

laboratory supervisor.
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-30

Table 7-3. Recommended Sample Aliquot Weights and Containers

for Various Analyses

Analysis Aliquot weight

(g)

Shipping/storage container

Metals 1-5 Polystyrene, borosilicate glass, or

PTFE jar with PTFE-lined lid

Organics 20-50 Glass or PTFE jar with PTFE-lined

lid

Dioxins/furans 20-50 Glass or PTFE jar with PTFE-lined

lid

PTFE = Polytetrafluoroethylene (Teflon).

The exact quantity of tissue required for each digestion or extraction and analysis

should be weighed and placed in an appropriate container that has been labeled

with the aliquot identification number, sample weight (to the nearest 0.1 g), and

the date aliquots were prepared (Stober, 1991). The analytical laboratory can

then recover the entire sample, including any liquid from thawing, by rinsing the

container directly into the digestion or extraction vessel with the appropriate

solvent. It is also the responsibility of the processing laboratory to provide a

sufficient number of aliquots for laboratory duplicates, matrix spikes, and matrix

spike duplicates so that the QC requirements of the program can be met (see

Sections 8.3.3.4 and 8.3.3.5), and to provide extra aliquots to allow for reanalysis

if the sample is lost or if QC control limits are not met.

It is essential that accurate records be maintained when aliquots are prepared for

analysis. Use of a carefully designed form is recommended to ensure that all the

necessary information is recorded. An example of a sample aliquot record is

shown in Figure 7-9. The composite sample identification number should be

assigned to the composite sample at the time of collection (see Section 6.2.3.1)

and carried through sample processing (plus "F1," "F2," or "C" if the composite

homogenate is comprised of individual or combined fillets). The aliquot

identification number should indicate the analyte class (e.g., MT for metals, OR

for organics, DX for dioxins) and the sample type (e.g., R for routine sample; RS

or a routine sample that is split for analysis by a second laboratory; MS1 and MS2

for sample pairs, one of which will be prepared as a matrix spike). For example,

the aliquot identification number may be WWWWW-XX-YY-ZZZ, where

WWWWW is a 5-digit sample composite identification number, XX indicates

individual (F1 or F2) or combined (C) fillets, YY is the analyte code, and ZZZ is the

sample type.

Blind laboratory duplicates should be introduced by preparing two separate

aliquots of the same composite homogenate and labeling one aliquot with a

"dummy" composite sample identification. However, the analyst who prepares the

laboratory duplicates must be careful to assign a "dummy" identification number

that has not been used for an actual sample and to indicate clearly on the
7-31Figure 7-9. Example of a fish and shellfish monitoring program sample aliquot record.
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-32

processing records that the samples are blind laboratory duplicates. The

analytical laboratory should not receive this information.

When the appropriate number of aliquots of a composite sample have been

prepared for all analyses to be performed on that sample, the remainder of the

composite sample should be labeled with "ARCHIVE" and the expiration date and

placed in a secure location at -20 C in the sample processing laboratory. The

location of the archived samples should be indicated on the sample aliquot record.

Unless analyses are to be performed immediately by the sample processing

laboratory, aliquots for sample analysis should be frozen at -20 C before they

are transferred or shipped to the appropriate analytical laboratory.

7.3.2 Sample Transfer

The frozen aliquots should be transferred on dry ice to the analytical laboratory

(or laboratories) accompanied by a sample transfer record such as the one shown

in Figure 7-10. Further details on federal regulations for shipping biological

specimens in dry ice are given in Section 6.3.3.2. The sample transfer record

may include a section that serves as the analytical laboratory COC record. The

COC record must be signed each time the samples change hands for preparation

and analysis.
7. LABORATORY PROCEDURES I — SAMPLE HANDLING

7-33

Date _____ _____ _____ Time ___:___ (24-h clock)

YYYY MM DD

Released by: 

(name)

At: 

(location)

Shipment Method 

Shipment Destination 

Date _____ _____ _____ Time ___:___ (24-h clock)

YYYY MM DD

Released by: 

(name)

At: 

(location)

Comments 

Study Type:  Screening — Analyze for:  Trace metals  Organics  Lipid

Intensive Phase 1  Phase II  — Analyze for (specify)

Sample IDs:

Laboratory Chain of Custody

Relinquished by Received by Purpose Location

Figure 7-10. Example of a fish and shellfish monitoring program sample transfer record.
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-1

SECTION 8

LABORATORY PROCEDURES II — SAMPLE ANALYSES

Sample analyses may be conducted by one or more state or private contract

laboratories. Because of the toxicity of dioxins/furans and the difficulty and cost

of these analyses, relatively few laboratories currently have the capability of

performing them. Table 8-1 lists contract laboratories experienced in dioxin/furan

analyses. This list is provided for information purposes only and is not an

endorsement of specific laboratories.

8.1 RECOMMENDED ANALYTES

8.1.1 Target Analytes

All recommended target analytes listed in Table 4-1 should be included in

screening studies unless reliable historic tissue, sediment, or pollutant source data

indicate that an analyte is not present at a level of concern for human health.

Additional target analytes should be included in screening studies if states have

site-specific information (e.g., historic tissue or sediment data, discharge

monitoring reports from municipal and industrial sources) that these contaminants

may be present at levels of concern for human health.

Intensive studies should include only those target analytes found to exceed

screening values in screening studies (see Section 5.2).

8.1.2 Lipid

A lipid analysis should also be performed and reported (as percent lipid by wet

weight) for each composite tissue sample in both screening and intensive studies.

This measurement is necessary to ensure that gel permeation chromatography

columns are not overloaded when used to clean up tissue extracts prior to

analysis of organic target analytes. In addition, because bioconcentration of

nonpolar organic compounds is dependent upon lipid content (i.e., the higher the

lipid content of the individual organism, the higher the residue in the organism),

lipid analysis is often considered essential by users of fish and shellfish monitoring

data. Consequently, it is important that lipid data are obtained for eventual

inclusion in a national database of fish and shellfish contaminant data.
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-2

Table 8-1. Contract Laboratories Conducting Dioxin/Furan Analyses In

Fish and Shellfish Tissuesa

Alta Analytical Laboratoryb

5070 Robert J. Matthews Parkway, Suite 2

Eldorado Hills, CA 95762 

916/933-1640

FAX: 916/933-0940

Bill Luksemburg

Battelle-Columbus Laboratoriesb

505 King Avenue

Columbus, OH 43201

614/424-7379

Karen Riggs

Midwest Research Instituteb

425 Volker Boulevard

Kansas City, MO 64110

816/753-7600, ext. 1160/1557

FAX: 816/753-8240

John Stanley/Tom Sack

e-mail: JStanley@mriresearch.org

tsack@mriresearch.org

New York State Department of Healthb

Wadsworth Center

Empire State Plaza

P.O. Box 509

Albany, NY 12201-0509

518/473-3378

FAX: 518/473-2895

Patrick O’Keefe

Pacific Analytical, Inc.b

6349 Paseo Del Argo

Carlsbad, CA 92009

760/438-3100

FAX: 760/931-9479

Bruce Colby

Axys Analytical Servicesb

P.O. Box 2219

2045 Mills Road

Sidney, BC V8L 3

Canada

250/656-0881; Toll Free 1-888-373-0881

Coreen Hamilton/Dale Hover/Laurie Phillips

Pace Analytical Servicesb

7726 Moller Road

Indianapolis, IN 46268

317/875-5894

FAX: 317/872-6189

Mick Mayse

Triangle Laboratoriesb

Alston Technical Park

801 Capitola Drive

Durham, NC 27713

919/544-5729

FAX: 919/544-5491

Phil Albro

Wellington Environmental Consultantsb

398 Laird Road

Guelph, Ontario N1G 3X7

Canada

519/822-2436

Judy Sparling/Brock Chittin/Colleen Tashiro

Wright State Universityb

175 Brehm Laboratory

3640 Colonel Glen Highway

Dayton, OH 45435

937/775-2202

FAX: 937/775-3122

Thomas Tiernan/Garrett Van Ness

Quanterra Environmental Services

Knoxville Laboratory

5815 Middlebrook Pike

Knoxville, TN 37921

423/588-6401

FAX: 423/584-4315

David Thal/Tom Yoder

a This list should not be construed as an endorsement by EPA of these laboratories, but is

provided for information purposes only. b Laboratory participating in Method 1613 interlaboratory (round-robin) dioxin study (May 1991).
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-3

Note: Because the concentrations of contaminants, particularly nonpolar

organics, are often correlated with the percentage of lipid in a tissue sample,

contaminant data are often normalized to the lipid concentration before statistical

analyses are performed. This procedure can, in some instances, improve the

power of the statistical tests. States wishing to examine the relationship between

contaminant concentrations and percentage of lipid should refer to Hebert and

Keenleyside (1995) for a discussion of the possible statistical approaches.

8.2 ANALYTICAL METHODS

This section provides guidance on selecting methods for analysis of recommended target analytes. Analytical methods should include appropriate

procedures for sample preparation (i.e., for digestion of samples to be analyzed

for metals and for extraction and extract cleanup of samples to be analyzed for

organics).

8.2.1 Lipid Method

It is recommended that a gravimetic method be used for lipid analysis. This

method is easy to perform and is commonly used by numerous laboratories,

employing various solvent systems such as chloroform/methanol (Bligh and Dyer,

1959), petroleum ether (California Department of Fish and Game, 1990; U.S.

FDA, 1990), and dichloromethane (NOAA, 1993a; Schmidt et al., 1985). The

results of lipid analyses may vary significantly (i.e., by factors of 2 or 3), however,

depending on the solvent system used for lipid extraction (Randall et al., 1991; D.

Swackhamer, University of Minesota, personal communication, 1993; D. Murphy,

Maryland Department of the Environment, Water Quality Toxics Division, personal

communication, 1993). Therefore, to ensure consistency of reported results

among fish contaminant monitoring programs, it is recommended that

dichloromethane be used as the extraction solvent in all lipid analyses. 

In addition to the effect of solvent systems on lipid analysis, other factors can also

increase the inter- and intralaboratory variation of results if not adequately

controlled (Randall et al., 1991). For example, high temperatures have been

found to result in decomposition of lipid material and, therefore, should be avoided

during extraction. Underestimation of total lipids can also result from denaturing

of lipids by solvent contaminants, lipid decomposition from exposure to oxygen or

light, and lipid degradation from changes in pH during cleanup. Overestimation of

total lipids may occur if a solvent such as alcohol is used, which results in

substantial coextraction of nonlipid material. It is essential that these potential

sources of error be considered when conducting and evaluating results of lipid

analyses. 
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-4

8.2.2 Target Analyte Methods

EPA has published interim procedures for sampling and analysis of priority

pollutants in fish tissue (U.S. EPA, 1981); however, official EPA-approved

methods are available only for the analysis of low parts-per-billion concentrations

of some metals in fish and shellfish tissues (U.S. EPA, 1991g). Because of the

lack of official EPA-approved methods for all recommended target analytes, and

to allow states and Regions flexibility in developing their analytical programs,

specific analytical methods for recommended target analytes in fish and shellfish

monitoring programs are not included in this guidance document. 

Note: A performance-based analytical program is recommended for the analysis

of target analytes. This recommendation is based on the assumption that the

analytical results produced by different laboratories and/or different methods will

be comparable if appropriate QC procedures are implemented within each

laboratory and if comparable analytical performance on round-robin comparative

analyses of standard reference materials or split sample analyses of field samples

can be demonstrated. This approach is intended to allow states to use costeffective procedures and to encourage the use of new or improved analytical

methods without compromising data quality. Performance-based analytical

programs currently are used in several fish and shellfish monitoring programs,

including the NOAA Status and Trends Program (Battelle, 1989b; Cantillo, 1991;

NOAA, 1987), the EPA Environmental Monitoring and Assessment Program

(EMAP) (U.S. EPA, 1991e), and the Puget Sound Estuary Program (1990d,

1990e).

Analytical methods used in fish and shellfish contaminant monitoring programs

should be selected using the following criteria:

• Technical merit—Methods should be technically sound; they should be

specific for the target analytes of concern and based on current, validated

analytical techniques that are widely accepted by the scientific community.

• Sensitivity—Method detection and quantitation limits should be sufficiently low

to allow reliable quantitation of the target analytes of concern at or below

selected screening values. Ideally, the method detection limit (in tissue)

should be at least five times lower than the selected SV for a given target

analyte (Puget Sound Estuary Program, 1990e).

• Data quality—The accuracy and precision should be adequate to ensure that

analytical data are of acceptable quality for program objectives.

• Cost-efficiency—Resource requirements should not be unreasonably high.

A review of current EPA guidance for chemical contaminant monitoring programs

and of analytical methods currently used or recommended in several of these

programs (as shown in Table 8-2) indicates that a limited number of analytical
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-5

Table 8-2. Current References for Analytical Methods for

Contaminants in Fish and Shellfish Tissues

• Analytical Chemistry of PCBs (Erickson, 1991)

• Analytical Methods for Pesticides and Plant Growth Regulators, Vol. 11 (Zweig and Sherma, 1980)

• Analytical Procedures and Quality Assurance Plan for the Determination of Mercury in Fish (U.S. EPA,

1989a)

• Analytical Procedures and Quality Assurance Plan for the Determination of Xenobiotic Chemical

Contaminants in Fish (U.S. EPA, 1989c)

• Analytical Procedures and Quality Assurance Plan for the Determination of PCDD/PCDF in Fish (U.S. EPA,

1989b)

• Arsenic Speciation by Coupling High-Performance Liquid Chromatography with Inductively Coupled Plasma

Mass Spectrometry (Demesmay et al., 1994)

• Assessment and Control of Bioconcentratable Contaminants in Surface Water (U.S. EPA, 1991a).

• Bioaccumulation Monitoring Guidance: 4. Analytical Methods for U.S. EPA Priority Pollutants and 301(h)

Pesticides in Tissues from Marine and Estuarine Organisms (U.S. EPA, 1986a)

• Determination of Arsenic Species by High-Performance Liquid Chromatography - Inductively Coupled

Plasma Mass Spectrometry (Beauchemin et al., 1989)

• Determination of Arsenic Species in Fish by Directly Coupled High-Performance Liquid ChromatographyInductively Coupled Plasma Mass Spectrometry (Branch et al., 1994)

• The quantitation of butyltin and cyclohexyltin compounds in the marine environment of British Columbia. 

Appl. Organometal. Chem. 4:581-590 (Cullen et al., 1990) 

• Determination of Butyltin, Methyltin and Tetraalkyltin in Marine Food Products with Gas ChromatographyAtomic Absorption Spectrometry (Forsyth and Cleroux, 1991)

• Determination of Tributyltin Contamination in Tissues by Capillary Column Gas Chromatography-Flame

Photometric Detection with Confirmation by Gas Chromatography-Mass Spectroscopy (Wade et al., 1988)

• Determination of Tributyltin in Tissues and Sediments by Graphite Furnace Atomic Absorption Spectrometry

(Stephenson and Smith, 1988) 

• Environmental Monitoring and Assessment Program Near Coastal Virginian Province Quality Assurance

Project Plan (Draft) (U.S. EPA, 1991e)

• Guidelines for Studies of Contaminants in Biological Tissues for the National Water-Quality Assessment

Program (Crawford and Luoma, 1993)

• Interim Methods for the Sampling and Analysis of Priority Pollutants in Sediments and Fish Tissue (U.S.

EPA, 1981)

• Laboratory Quality Assurance Program Plan (California Department of Fish and Game, 1990)

• Methods for Organic Analysis of Municipal and Industrial Wastewater (40 CFR 136, Appendix A).

• Methods for the Chemical Analysis of Water and Wastes (U.S. EPA, 1979b)

• Methods for the Determination of Metals in Environmental Samples (U.S. EPA, 1991g)

• Official Methods of Analysis of the Association of Official Analytical Chemists (Williams, 1984)

• Pesticide Analytical Manual (PAM Vols. I and II) (U.S. FDA, 1990)

• Puget Sound Estuary Program Plan (1990d, 1990e)

• Quality Assurance/Quality Control (QA/QC) for 301(h) Monitoring Programs: Guidance on Field and

Laboratory Methods (U.S. EPA, 1987e)

(continued)
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-6

Table 8-2 (continued)

• Sampling and Analytical Methods of the National Status and Trends Program National Benthic

Surveillance and Mussel Watch Projects 1984-92. Volume II. Comprehensive Descriptions of

Complementary Measurements (NOAA, 1993a)

• Sampling and Analytical Methods of the National Status and Trends Program National Benthic

Surveillance and Mussel Watch Projects 1984-92. Volume III. Comprehensive Descriptions of Elemental

Analytical Methods (NOAA, 1993b)

• Sampling and Analytical Methods of the National Status and Trends Program National Benthic

Surveillance and Mussel Watch Projects 1984-92. Volume IV. Comprehensive Descriptions of Trace

Organic Analytical Methods (NOAA, 1993c)

• Separation of Seven Arsenic Compounds by High-performance Liquid Chromatography with On-line

Detection by Hydrogen-Argon Flame Atomic Absorption Spectrometry and Inductively Coupled Plasma

Mass Spectrometry (Hansen et al., 1992)

• Speciation of Selenium and Arsenic in Natural Waters and Sediments by Hydride Generation Followed by

Atomic Absorption Spectroscopy (Crecelius et al., 1986)

• Standard Analytical Procedures of the NOAA National Analytical Facility (Krahn et al., 1988; MacLeod et

al., 1985)

• Standard Methods for the Examination of Water and Wastewater (Greenburg et al., 1992)

• Test Methods for the Chemical Analysis of Municipal and Industrial Wastewater (U.S. EPA, 1982)

• Test Methods for the Evaluation of Solid Waste, Physical/Chemical Methods (SW-846) (U.S. EPA, 1986d)

• U.S. EPA Contract Laboratory Program Statement of Work for Inorganic Analysis (U.S. EPA, 1991b)

• U.S. EPA Contract Laboratory Program Statement of Work for Organic Analysis (U.S. EPA, 1991c)

• U.S. EPA Method 1613B: Tetra- through Octa-Chlorinated Dioxins and Furans by Isotope Dilution

HRGC/HRMS (U.S. EPA, 1995b)

• U.S. EPA Method 1625: Semivolatile Organic Compounds by Isotope Dilution GC/MS (40 CFR 136,

Appendix A)

• U.S. EPA Method 1631: Mercury in Water by Oxidation, Purge and Trap, and Cold Vapor Atomic

Fluorescence Spectrometry (U.S. EPA, 1995c)

• U.S. EPA Method 1632: Determination of Inorganic Arsenic in Water by Hydride Generation Flame

Atomic Absorption (U.S. EPA, 1995d)

• U.S. EPA Method 1637: Determination of Trace Elements in Ambient Waters by Chelation

Preconcentration with Graphite Furnace Atomic Absorption (U.S. EPA, 1995e)

• U.S. EPA Method 1638: Determination of Trace Elements in Ambient Waters by Inductively Coupled

Plasma-Mass Spectrometry (U.S. EPA, 1995f)

• U.S. EPA Method 1639: Determination of Trace Elements in Ambient Waters by Stabilized Temperature

Graphite Furnace Atomic Absorption (U.S. EPA, 1995g)

• U.S. EPA Method 625: Base/Neutrals and Acids by GC/MS (40 CFR 136, Appendix A).

• U.S. EPA Method 8290: Polychlorinated Dibenzodioxins (PCDDs) and Polychlorinated Dibenzofurans

(PCDFs) by High Resolution Gas Chromatography/High Resolution Mass Spectrometry (HRGC/HRMS)

(U.S. EPA, 1990b)

• U.S. EPA Method 1668: Draft Method 1668 Toxic Polychlorinated Biphenols by Isotope Dilution High Gas

Chromatography/High Resolution Mass Spectrometry (U.S. EPA, 1997a)
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-7

techniques are most commonly used for the determination of the recommended

target analytes. These techniques are listed in Table 8-3. As shown in Table 8-4,

analytical methods employing these techniques have typically achievable

detection and/or quantitation limits that are well below the recommended SVs for

most target analytes, with the possible exception of dieldrin, heptachlor epoxide,

toxaphene, PCBs, and dioxins/furans. Recommended procedures for determining

method detection and quantitation limits are given in Section 8.3.3.3.

If lower SVs are used in a study (e.g., for susceptible populations), it is the

responsibility of program managers to ensure that the detection and quantitation

limits of the analytical methods are sufficiently low to allow reliable quantitation of

target analytes at or below these SVs. If analytical methodology is not sensitive

enough to reliably quantitate target analytes at or below selected SVs (e.g.,

dieldrin, heptachlor epoxide, toxaphene, PCBs, dioxins/furans), program

managers must determine appropriate fish consumption guidance based on

lowest detectable concentrations or provide justification for adjusting SVs to

values at or above achievable method detection limits. It should be emphasized

that when SVs are below detection limits, the failure to detect a target analyte

cannot be assumed to mean that there is no cause for concern for human health

effects.

The analytical techniques identified in Table 8-3 are recommended for use in state

fish and shellfish contaminant monitoring programs. However, alternative

techniques may be used if acceptable detection limits, accuracy, and precision

can be demonstrated. Note: Neither rotenone, the most widely used piscicide in

the United States, nor its biotransformation products (e.g., rotenolone, 6’,7’-

dihydro-6’,7’-dihydroxyrotenone, 6’,7’-dihydro-6’,7’-dihydroxyrotenolone) would be

expected to interfere with the analyses of organic target analytes using the

recommended gas chromatographic methods of analysis. Furthermore, rotenone

has a relatively short half-life in water (3.7, 1.3, and 5.2 days for spring, summer,

and fall treatments, respectively) (Dawson et al., 1991) and does not bioaccumulate significantly in fish (bioconcentration factor= 26 in fish carcass) (Gingerich

and Rach, 1985), so that tissue residues should not be significant. 

Laboratories should select analytical methods for routine analyses of target

analytes that are most appropriate for their programs based on available

resources, experience, program objectives, and data quality requirements. A

recent evaluation of current methods for the analyses of organic and trace metal

target analytes in fish tissue provides useful guidance on method selection,

validation, and data reporting procedures (Capuzzo et al., 1990).

The references in Table 8-2 should be consulted in selecting appropriate analytical methods. Note: Because many laboratories may have limited experience in

determining inorganic arsenic, a widely accepted method for this analysis is

included in Appendix H. 
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-8

Table 8-3. Recommended Analytical Techniques for Target Analytes

Target analyte Analytical technique

Metals

Arsenic (inorganic) HAA, or HPLC with ICP-MS

Cadmium GFAA or ICPa

Mercury CVAA

Selenium GFAA, ICP, or HAAa,b

Tributyltin GFAA or GC/FPDc

Organics

PAHs GC/MS or HRGC/HRMSd

PCBs

Total Aroclors GC/ECDe,f,g,h

Non-ortho coplanar PCBs HRGC/HRMSi

Other cogeners/homologs HRGC/LRMS

Organochlorine pesticides GC/ECDf,g

Organophosphate pesticides GC/MS, GC/FPD, or GC/NPDj

Chlorophenoxy herbicides GC/ECDf,g

Dioxins/dibenzofurans HRGC/HRMSk,l

CVAA = Cold vapor atomic absorption spectrophotometry.

GC/ECD = Gas chromatography/electron capture detection.

GC/FPD = Gas chromatography/flame photometric detection.

GC/MS = Gas chromatography/mass spectrometry.

GC/NPD = Gas chromatography/nitrogen-phosphorus detection.

GFAA = Graphite furnace atomic absorption spectrophotometry.

HAA = Hydride generation atomic absorption spectrophotometry.

HPLC = High-performance liquid chromatography.

HRGC/HRMS = High-resolution gas chromatography/high-resolution mass spectrometry.

ICP = Inductively coupled plasma emission spectrometry.

ICP-MS = Inductively coupled plasma mass spectrometry.

LRMS = Low resolution mass spectrometry.

PAHs = Polycyclic aromatic hydrocarbons. 

PCBs = Polychlorinated biphenyls. a Atomic absorption methods require a separate determination for each element, which increases the time and cost relative

to the broad-scan ICP method. However, GFAA detection limits are typically more than an order of magnitude lower than

those achieved with ICP. b Use of HAA can lower detection limits for selenium by a factor of 10-100 (Crecelius, 1978; Skoog, 1985). c GC/FDP is specific for tributyltin and the most widely accepted analytical method. GFAA is less expensive (see Table 8-5)

but is not specific for tributyltin. Depending on the extraction scheme, mono-, di-, and tetrabutyltin and other alkyltins may

be included in the analysis. Contamination of samples with tin may also be a potential problem, resulting in false positives

(E. Crecelius, Battelle Pacific Northwest Laboratories, Marine Sciences Laboratory, Sequim, WA, personal

communication, 1999). d GC/MS is also recommended for base/neutral organic target analytes (except organochlorine pesticides and PCBs) that

may be included in a study. Detection limits of less than 1 ppb can be achieved for PAHs using HRGC/HRMS. It is

recommended that, in both screening and intensive studies, tissue samples be analyzed for benzo[a]pyrene and 14 other

PAHs and that the relative potencies given for these PAHs (Nesbit and LaGoy, 1992; U.S. EPA, 1993c) be used to

calculate PEC for each sample for comparison with the recommended SV for benzo[a]pyrene (see Section 5.3.2.4). e Analysis of total PCBs, as the sum of PCB congeners or sum of Aroclors, is recommended for conducting human health

risk assessments for PCBs. A standard method for Aroclor analyses is available (EPA Method 608). EPA is currently

testing a draft method (1668) for PCB congener analysis; however, it has not been finalized.

(continued)
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-9

Table 8-3 (continued)

f GC/ECD does not provide definitive compound identification, and false positives due to interferences are commonly reported.

Confirmation by an alternative GC column phase (with ECD), or by GC/MS with selected ion monitoring, is required for positive

identification of PCBs, organochlorine pesticides, and chlorophenoxy herbicides. g GC/MS with selected ion monitoring may be used for quantitative analyses of these compounds if acceptable detection limits can

be achieved. h PCB congener analysis using capillary GC columns is recommended (NOAA, 1989b; Dunn et al., 1984; Schwartz et al., 1984; Mullin

et al., 1984; Stalling et al., 1987). An enrichment step, employing an activated carbon column, may also be required to separate and

quantify coeluting congeners or congeners present at very low concentrations (Smith, 1981; Schwartz et al., 1993). i Includes PCBs -77, -81, -126 and -169. j Some of the chlorinated organophosphate pesticides (e.g., chlorpyrifos) may be analyzed by GC/ECD (USGS, 1987). k The analysis of the 17 2,3,7,8-substituted congeners of tetra- through octa-chlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans

(PCDFs) using isotope dilution is recommended. l Because of the toxicity of dioxins/furans and the difficulty and cost of these analyses, relatively few laboratories currently have the

capability of performing these analyses. Contract laboratories experienced in conducting dioxin/furan analyses are listed in Table

8-1.
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-10

Table 8-4. Range of Detection and Quantitation Limits of Current Analytical Methods

for Recommended Target Analytesa

Target analyte

Recreational

SV

Subsistence

SVb Range of detection limits

Range of

quantitation

limits

Metals

Arsenic (inorganic)

Cadmium

26 ppb

4,000 ppb

3.27 ppb

491 ppb

5-50 ppbc

; 50-100 ppbd

5-46 ppbe, 400 ppbf 5-25 ppb

5-500 ppb

Mercury

Selenium

400 ppb

20,000 ppb

49 ppb

2,457 ppb

1.3-100 ppbg

17-150 ppbc; 20 ppbh, 600 ppbf 2-10 ppb

20-600 ppb

Tributyltin 1,200 ppb 147 ppb 2.5 ppbe

; 2-5 ppbi 2-10 ppb

Organochlorine Pesticidesj

Chlordane (total)

cis-Chlordane

trans-Chlordane

cis-Nonachlor

trans-Nonachlor

Oxychlordane

—

114 ppb

—

14 ppb 1-5 ppb

1-5 ppb

1-5 ppb

1-7 ppb

1-5 ppb

2-20 ppbj,k

2-15 ppb

2-15 ppb

2-15 ppb

2-15 ppb

DDT (total)

4,4 ́-DDT

2,4 ́-DDT

4,4 ́-DDD

2,4 ́-DDD

4,4 ́-DDE

2,4 ́-DDE

117 ppb 14.4 ppb

0.1-13 ppb

0.1-10 ppb

0.1-10 ppb

0.1-10 ppb

0.1-38 ppb

0.1-10 ppb

2-20 ppb

2-15 ppb

2-15 ppb

2-15 ppb

2-15 ppbj,k

2-15 ppb

Dicofol 1,600 ppb 196 ppb 1-5 ppb 2-10 ppb

Dieldrin 2.50 ppb 0.307 ppb 0.1-5 ppb 2-15 ppb

Endosulfan (total)

Endosulfan I

Endosulfan II

Endrin

Heptachlor epoxide

Hexachlorobenzene

Lindane

Mirex

Toxaphene

24,000 ppb

1,200 ppb

4.39 ppb

25 ppb

30 ppb

800 ppb

36 ppb

2,949 ppb

147 ppb

0.54 ppb

3.07 ppb

3.78 ppb

98 ppb

4.46 ppb

5-70 ppb

5-10 ppb

5-70 ppb

0.1-15 ppb

0.1-5 ppb

0.1-2 ppb

0.1-5 ppb

0.1-5 ppb

3-100 ppb

10-70 ppb

2-15 ppb

10-70 ppb

2-15 ppbj,k

2-15 ppbj,k

2-15 ppbj,k

2-15 ppbj,k

2-15 ppbj,k

60-153 ppb

Organophosphate Pesticidesj

 Chlorpyrifos

 Diazinon

 Disulfoton

 Ethion

 Terbufos

18,000 ppb

4,200 ppb

240 ppb

3,000 ppb

120 ppb

147 ppb

344 ppb

19 ppb

245 ppb

9 ppb

2-5 ppb

2-5 ppb

2-5 ppb

2-5 ppb

2-5 ppb

2-15 ppbk

2-15 ppb

2-15 ppb

2-15 ppb

2-15 ppb

Chlorophenoxy Herbicides

 Oxyfluorfen 546 ppb 679 ppb 10-20 ppb 20-200 ppb

PAHsl 5.47 ppb 0.67 ppb 1-10 ppt 2-20 ppt

PCBs total

(sum of Aroclors)j

Non-ortho coplanar PCBsk

Other congeners/ homologuesn

20 ppb 2.45 ppb —

(20-62 ppb)m

2-5 ppt

2-5 ppb

—

(110-170 ppb)m

2-10 ppt

10 ppb

Dioxins/furansk

 (total)

 TCDD/TCDF

 PeCDD/PeCDF

 HxCDD/HxCDF

 HpCDD/HpCDF

 OCDD/OCDF

0.256 ppt 0.031 ppt 1.0 ppt

0.1 ppt

0.5 ppt

0.5 ppt

0.5 ppt

1.0 ppt

5-10 ppt

0.5 ppt

2.5 ppt

2.5 ppt

2.5 ppt

5 ppt

PAHs = Polycyclic aromatic hydrocarbons. PCBs = Polychlorinated biphenyls. SV = Screening value (wet weight).

(continued)
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-11

An additional resource for method selection is the EPA Environmental Monitoring

Methods Index System (EMMI), an automated inventory of information on

environmentally significant analytes and methods for their analysis (U.S. EPA,

1991f). The EMMI database includes information on more than 4,000 analytes

from over 80 regulatory and nonregulatory lists and more than 900 analytical

methods in a variety of matrices, including tissue. This searchable database

provides a comprehensive cross-reference between analytes and analytical

methods with detailed information on each analytical method, including

sponsoring organization, sample matrix, and estimates of detection limits,

accuracy, and precision.

EMMI is available from the EPA Sample Control Center for all EPA personnel and

from National Technical Information Service (NTIS) for all other parties. EMMI is

also available through the EPA Local Area Network (LAN).

Table 8-4. (continued)

a Target analyte concentrations are given based on wet weight of fish tissue. b From Tables 5-2 and 5-3. SVs shown here are for fish consumers using RfDs or CSFs available in the EPA IRIS

(1999) database and assuming a consumption rate (CR) for recreational fishers of 12 g/d and for subsistence

fishers of 124 g/d , average adult body weight (BW) = 72 kg, lifetime (70-yr) exposure, and, for carcinogens, a risk

level (RL) = 10-5. Note: Increasing CR, decreasing BW, and/or using an RL <10-5 will decrease the SV. Program

managers must ensure that detection and quantitation limits of analytical methods are sufficient to allow reliable

quantitation of target analytes at or below selected SVs. If analytical methodology is not sensitive enough to

reliably quantitate target analytes at or below selected SVs (e.g., inorganic arsenic, dieldrin, heptachlor epoxide,

toxaphene, PCBs, dioxins/furans), the program managers must determine appropriate fish consumption guidance

based on lowest detectable concentrations or provide justification for adjusting SVs to values at or above

achievable method detection or quantitation limits. It should be emphasized that when SVs are below method

detection limits, the failure to detect a target analyte cannot be assumed to indicate that there is no cause for

concern for human health effects. c Analysis by hydride generation atomic absorption spectrophotometry (HAA) with preconcentration (E. Crecelius,

Battelle Pacific Northwest Laboratories, Marine Sciences Laboratory, Sequim, WA, personal communication, July

1999). d Analysis by high-performance liquid chromatography/mass spectrometry (HPLC/MS) (E. Crecelius, Battelle Pacific

Northwest Laboratories, Marine Sciences Laboratory, Sequim, WA, personal communication, July 1999). e Analysis by graphite furnace atomic absorption spectrophotometry (GFAA). Note: This method is not specific for

tributyltin. Depending on the extraction procedure, mono-, di-, and tetrabutyltin may also be included in the

analysis. Also, this method does not distinguish between butyltins and other alkyltins (E. Crecelius, Battelle Pacific

Northwest Laboratories, Marine Sciences Laboratory, Sequim, WA, personal communication, July 1999). f Analysis by inductively coupled plasma atomic emission spectrophotometry (ICP). g Analysis by cold vapor atomic absorption spectrophotometry (CVAA). h Analysis by HAA. i Analysis by gas chromatography/flame photometric detection (GC/FPD) (E. Crecelius, Battelle Pacific Northwest

Laboratories, Marine Sciences Laboratory, Sequim, WA, personal communication, July 1999).

j Analysis by gas chromatography/electron capture detection (GC/ECD), except where otherwise noted. k Analysis by high-resolution GC/high-resolution mass spectrometry (HRGC/HRMS). l Analysis by gas chromatography/mass spectrometry. Detection limits of less than 1 ppb can be achieved using

high-resolution gas chromatography/mass spectrometry (HRGC/HRMS). m Values in parentheses represent ranges for individual Aroclors. n Analysis by high-resolution GC/low resolution mass spectrometry (HRGC/LRMS).
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-12

The private sector may purchase EMMI Version 2.0 through the:

National Technical Information Service (NTIS)

5285 Port Royal Road

Springfield, VA 22161

USA

Phone: (703) 605-6000

Fax: (703) 605-6900

Rush Orders: (800) 553-NTIS

Online Orders: http:\\www.ntis.gov

The order number is PB97-5026371NC for a single user, PB97-502645INC for a

five-user LAN package, and PB97-502652INC for an unlimited user LAN package.

Further information may be obtained by contacting:

EMMI User Support

Tech Calls

EPA Assistant Administrator for Water 

Office of Science and Technology

(703) 461-2104

Alexandria, VA 22313

Because chemical analysis is frequently one of the most expensive components

of a sampling and analysis program, the selection of an analytical method often

will be influenced by its cost. In general, analytical costs increase with increased

sensitivity (i.e., lower detection limits) and reliability (i.e., accuracy and precision).

Analytical costs will also be dependent on the number of samples to be analyzed,

the requested turnaround time, the number and type of analytes requested, the

level of QC effort, and the amount of support documentation requested (Puget

Sound Estuary Program, 1990d). However, differences in protocols, laboratory

experience, and pricing policies of laboratories often introduce large variation into

analytical costs. Approximate costs per sample for the analysis of target analytes

by the recommended analytical techniques are provided in Table 8-5.

8.3 QUALITY ASSURANCE AND QUALITY CONTROL CONSIDERATIONS

Quality assurance and quality control must be integral parts of each chemical

analysis program. The QA process consists of management review and oversight

at the planning, implementation, and completion stages of the analytical data

collection activity to ensure that data provided are of the quality required. The QC

process includes those activities required during data collection to produce the

data quality desired and to document the quality of the collected data.

During the planning of a chemical analysis program, QA activities focus on

defining data quality criteria and designing a QC system to measure the quality

of data being generated. During the implementation of the data collection effort,

QA activities ensure that the QC system is functioning effectively and that the
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-13

Table 8-5. Approximate Range of Costs per Sample for 

Analysis of Recommended Target Analytesa

Target analyte Approximate cost range (1999 $)

Metalsb

Arsenic (inorganic)c

Cadmium 

Mercury (total)

Selenium 

Tributyltind

200 - 400 

55 - 60 

45 - 60

35 - 60

200 - 400

Organochlorine pesticidese,f

Organophosphate pesticidesg

Chlorophenoxy herbicidesh

285 - 500

250 - 500

250 - 500

PAHsi 250 - 525

PCBs

Total Aroclorse

Non-ortho coplanar PCBsj

Other cogeners/homologsk

210 - 500

1,000 - 2,000

800 - 1,000

Dioxins/furansj

TCDD/TCDF only

TCDD/TCDF through OCDD/OCDF isomers

2,3,7,8-substituted dioxins/furans

600 - 1,000

800 - 1,600

1,000 - 2,000

Lipid 30 - 40

OCDD = Octachlorodibenzo-p-dioxin.

OCDF = Octachlorodibenzofuran.

PAHs = Polycyclic aromatic hydrocarbons.

PCBs = Polychlorinated biphenyls.

TCDD = 2,3,7,8-Tetrachlorodibenzo-p-dioxin.

TCDF = 2,3,7,8-Tetrachlorodibenzofuran. a These costs include sample digestion or extraction and cleanup, but not sample preparation (i.e., resection,

grinding, homogenization, compositing). Estimated cost of sample preparation for a composite homogenate

of five fish is $200 to $500. b Analysis of inorganic arsenic by hydride generation atomic absorption spectroscopy (HAA) or highperformance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP/MS). 

Analysis of cadmium by graphite furnace atomic absorption spectrophotometry (GFAA). Analysis of

selenium by GFAA or HAA. Analysis of mercury by cold vapor atomic absorption spectrophotometry (CVAA). 

Analysis of tributyltin by GFAA or gas chromatography/flame photometric detection (GC/FPD). c Estimated costs are for total inorganic arsenic. Estimated cost of analysis by HAA is $200. Estimated cost

of analysis by HPLC-ICP/MS is $400. d Estimated cost of analysis by GFAA is $200. Estimated cost of analysis by GC/FPD is $400. Note:

Analysis by GFAA is not specific for tributyltin. Depending on the extraction procedure, other butyl- and

alkyltin species may be detected. e Analysis by gas chromatography/electron capture detection (GC/ECD). f Estimated costs are for analysis of all recommended target analyte organochlorine pesticides (see

Table 4-1). g Analysis by GC/FPD or gas chromatography/nitrogen-phosphorus detection (GC/NPD). Some of the

chlorinated organophosphate pesticides (e.g., chlorpyrifos) may be analyzed as organochlorine pesticides

by GC/ECD (USGS, 1987). h Analysis by GC/ECD. i Costs are for analysis by gas chromatography/mass spectrometry (GC/MS) or gas chromatography/flame

ionization detection (GC/FID). Cost for analysis by high-resolution gas chromatography/high resolution

mass spectrometry (HRGC/HRMS) is approximately $1,000 per sample.

j Analysis by HRGC/HRMS. k Analysis by HRGC/low resolution mass spectrometry (LRMS).
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-14

deficiencies uncovered by the QC system are corrected. After the analytical data

are collected, QA activities focus on assessing the quality of data obtained to

determine its suitability to support decisions for further monitoring, risk

assessments, or issuance of advisories.

The purpose of this section is to describe the general QA and QC requirements

for chemical analysis programs.

8.3.1 QA Plans

Each laboratory performing chemical analyses in fish and shellfish contaminant

monitoring programs must have an adequate QA program (U.S. EPA, 1984b).

The QA program should be documented fully in a QA plan or in a combined

Work/QA Project Plan (U.S. EPA, 1980b). (See Appendix I.) Each QA and QC

requirement or procedure should be described clearly. Documentation should

clearly demonstrate that the QA program meets overall program objectives and

data quality requirements. The QA guidelines in the Puget Sound Estuary

Program (1990d, 1990e), the NOAA Status and Trends Program (Battelle, 1989b;

Cantillo, 1991; NOAA, 1987), the EPA 301(h) Monitoring Programs (U.S. EPA,

1987e), the EPA EMAP Near Coastal (EMAP-NC) Program (U.S. EPA, 1991e),

and the EPA Contract Laboratory (CLP) Program (U.S. EPA, 1991b, 1991c) are

recommended as a basis for developing program-specific QA programs.

Additional method-specific QC guidance is given in references in Table 8-2.

8.3.2 Method Documentation

Methods used routinely for the analyses of contaminants in fish and shellfish

tissues must be documented thoroughly, preferably as formal standard operating

procedures (U.S. EPA, 1984b). Recommended contents of an analytical SOP are

shown in Figure 8-1. Analytical SOPs must be followed exactly as written. A

published method may serve as an analytical SOP only if the analysis is

performed exactly as described. Any significant deviations from analytical SOPs

must be documented in the laboratory records (signed and dated by the

responsible person) and noted in the final data report. Adequate evidence must

be provided to demonstrate that an SOP deviation did not adversely affect method

performance (i.e., detection or quantitation limits, accuracy, precision). Otherwise, the effect of the deviation on data quality must be assessed and

documented and all suspect data must be identified.

8.3.3 Minimum QA and QC Requirements for Sample Analyses

The guidance provided in this section is derived primarily from the protocols

developed for the Puget Sound Estuary Program (1990d, 1990e). These

protocols have also provided the basis for the EPA EMAP-NC QA and QC

requirements (U.S. EPA, 1991e). QA and QC recommendations specified in this

document are intended to provide a uniform performance standard for all

analytical protocols used in state fish and shellfish contaminant monitoring
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-15

• Scope and application

• Method performance characteristics (accuracy,

precision, method detection and quantitation limits)

for each analyte

• Interferences

• Equipment, supplies, and materials

• Sample preservation and handling procedures

• Instrument calibration procedures

• Sample preparation (i.e., extraction, digestion,

cleanup) procedures

• Sample analysis procedures

• Quality control procedures

• Corrective action procedures

• Data reduction and analysis procedures (with

example calculations)

• Recordkeeping procedures (with standard data

forms, if applicable)

• Safety procedures and/or cautionary notes

• Disposal procedures

• References

Figure 8-1. Recommended contents of analytical standard operating procedures (SOPs).

programs and to enable an assessment of the comparability of results generated

by different laboratories and different analytical procedures. These recommendations are intended to represent minimum QA and QC procedures for any given

analytical method. Additional method-specific QC procedures should always be

followed to ensure overall data quality.

For sample analyses, minimum QA and QC requirements consist of (1) initial

demonstration of laboratory capability and (2) routine analyses of appropriate QA

and QC samples to demonstrate continued acceptable performance and to

document data quality.

Initial demonstration of laboratory capability (prior to analysis of field samples)

should include

• Instrument calibration

• Documentation of detection and quantitation limits

• Documentation of accuracy and precision

• Analysis of an accuracy-based performance evaluation sample provided by

an external QA program.

Ongoing demonstration of acceptable laboratory performance and documentation

of data quality should include

• Routine calibration and calibration checks 

• Routine assessment of accuracy and precision

• Routine monitoring of interferences and contamination

• Regular assessment of performance through participation in external QA

interlaboratory comparison exercises, when available.

The QA and QC requirements for the analyses of target analytes in tissues should

be based on specific performance criteria (i.e., warning or control limits) for data

quality indicators such as accuracy and precision. Warning limits are numerical

criteria that serve to alert data reviewers and data users that data quality may be

questionable. A laboratory is not required to terminate analyses when a warning

limit is exceeded, but the reported data may be qualified during subsequent QA

review. Control limits are numerical data criteria that, when exceeded, require
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-16

suspension of analyses and specific corrective action by the laboratory before the

analyses may resume.

Typically, warning and control limits for accuracy are based on the historical mean

recovery plus or minus two or three standard deviation units, respectively.

Warning and control limits for precision are typically based on the historical

standard deviation or coefficient of variation (or mean relative percent difference

for duplicate samples) plus two or three standard deviation units, respectively.

Procedures incorporating control charts (ASTM, 1976; Taylor, 1985) and/or

tabular presentations of historical data should be in place for routine monitoring

of analytical performance. Procedures for corrective action in the event of

excursion outside warning and control limits should also be in place.

The results for the various QC samples analyzed with each batch of samples

should be reviewed by qualified laboratory personnel immediately following the

analysis of each sample batch to determine when warning or control limits have

been exceeded. When established control limits are exceeded, appropriate

corrective action should be taken and, if possible, all suspect samples reanalyzed

before resuming routine analyses. If reanalyses cannot be performed, all suspect

data should be identified clearly. Note: For the purposes of this guidance

manual, a batch is defined as any group of samples from the same source that

is processed at the same time and analyzed during the same analytical run.

Recommended QA and QC samples (with definitions and specifications),

frequencies of analyses, control limits, and corrective actions are summarized in

Table 8-6.

Note: EPA recognizes that resource limitations may prevent some states from

fully implementing all recommended QA and QC procedures. Therefore, as

additional guidance, the minimum numbers of QA and QC samples recommended

for routine analyses of target analytes are summarized in Table 8-7. It is the

responsibility of each program manager to ensure that the analytical QC program

is adequate to meet program data quality objectives for method detection limits,

accuracy, precision, and comparability.

Recommended QA and QC procedures and the use of appropriate QA and QC

samples are discussed in Sections 8.3.3.2 through 8.3.3.8. Recommended

procedures for documenting and reporting analytical and QA and QC data are

given in Section 8.4. Because of their importance in assessing data quality and

interlaboratory comparability, reference materials are discussed separately in the

following section.
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-17(continued)

Table 8-6. Recommended Quality Assurance and Quality Control Samples

Sample type

(definition;

specifications) Objective

Recommended

frequency of analysisa

Recommended

control limitsb

Recommended 

corrective action

External CalibrationCalibration standards(3-5 standards over the expectedrange of sample target analyteconcentrations, with the lowestconcentration standard at or nearthe MDL; see Section 8.3.3.2.1)

Full calibration: Establishrelationship betweeninstrument response andtarget analyteconcentration. Used fororganics analysis byGC/ECD and for metalsanalysis.

Instrument/method-dependent; followmanufacturer’s recommendations orprocedures in specific analyticalprotocols. At a minimum, perform a 3-point calibration each time an instrumentis set up for analysis, after each majorequipment change or disruption, andwhen routine calibration check exceedsspecific control limits.

Organics: RSD of RFs of calibration standards <20%.

Metals: %R of all calibration standards = 95-105.

Determine cause of problem (e.g.,instrument instability or malfunction,contamination, inaccuratepreparation of calibrationstandards) and take appropriatecorrective action. Recalibrate andreanalyze all suspect samples orflag all suspect data.

Internal Standard CalibrationInstrument internalstandards (e.g., 2,2’-

difluorobiphenyl)(see Section 8.3.3.2.1 fordefinition)

Full calibration: DetermineRRFs of organic targetanalytes for quantitativeanalysis. Required forinternal calibration ofGC/MS systems. Optionalcalibration technique forGC/ECD.

In every calibration standard, sample,and blank analyzed; added to finalsample extract. Internal standardcalibration performed at same frequencyrecommended for external calibration.

RSD of RRFs of calibration standards

#30%.

Determine cause of problem (e.g.,instrument instability or malfunction,contamination, inaccuratepreparation of internal standards orcalibration standards) and takeappropriate corrective action. Recalibrate and reanalyze allsuspect samples or flag all suspectdata.

Calibration VerificationCalibration check standards(minimum of one mid-rangestandard prepared independentlyfrom initial calibration standards;an instrument internal standardmust be added to eachcalibration check standard wheninternal standard calibration isbeing used; see Section8.3.3.2.1)

Verify calibration. Organics (GC/MS): After initial

calibration or recalibration. Atbeginning and end of each work shift,and once every 12 h (or every 10-12analyses, whichever is morefrequent).

Organics (GC/ECD): After initial

calibration or recalibration. Atbeginning and end of each work shift,and once every 6 h (or every 6samples, whichever is less frequent).

Metals: After initial calibration or

recalibration. Every 10 samples orevery 2 h, whichever is more frequent.

Organics: Percent difference

between the average RF (or RRF)from initial calibration and the RF(or RRF) from the calibration check<25%.

Mercury: %R = 80-120.Other Metals: %R = 90-110.

Determine cause of problem (e.g.,instrument instability or malfunction,contamination, inaccuratepreparation of calibrationstandards) and take appropriatecorrective action. Recalibrate andreanalyze all suspect samples orflag all suspect data.
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-18

Table 8-6. (continued)

Sample type

(definition;

specifications) Objective

Recommended

frequency of analysisa

Recommended

control limitsb

Recommended 

corrective action

(continued)

Method Detection Limit DeterminationSpiked matrix samples(analyte-free tissue samples towhich known amounts of targetanalytes have been added; onespike for each target analyte at 3-5 times the estimated MDL; seeSection 8.3.3.3.1)

Establish or confirm MDLfor analyte of interest(Keith, 1991a; Keith et al.,1983).

Seven replicate analyses prior to use ofmethod for routine analyses, and afterany significant change to a methodcurrently in use. Reevaluation of MDLannually.

Determined by program manager. Redetermine MDL.

Accuracy and Precision AssessmentReference materialsc(see Section 8.3.3.1 fordefinitions)(SRMs or CRMs, prepared fromactual contaminated fish orshellfish tissue if possible, covering the range of expectedtarget analyte concentrations. 

Assess methodperformance (initialmethod validation androutine accuracyassessment).

Method validation: As many as requiredto assess accuracy (and precision) ofmethod before routine analysis ofsamples (i.e., when using a method forthe first time or after any methodmodification).Routine accuracy assessment: one

(preferably blind) per 20 samples or oneper batch, whichever is more frequent.

Organics: Measured value 

<95% confidence intervals, ifcertified. Otherwise, %R = 70-130.d

Metals: %R = 85-115.dOrganics: Measured value

<95% confidence intervals, ifcertified. Otherwise, %R = 70-130.d

Metals: %R = 85-115.d

NA

Determine cause of problem (e.g.,inaccurate calibration,contamination), take appropriatecorrective action, and reanalyze allsuspect samples or flag all suspectdata.

Laboratory control samples(Accuracy-based samplesconsisting of fish or shellfishtissue homogenates spiked withtarget analytes of interest; maybe SRMs or CRMs; sometimesreferred to as QC samples. When available, CRMs arerecommended for routine use aslaboratory control samples; seeAppendix M)

Assess methodperformance (initialmethod validation androutine accuracyassessment). Used forinitial accuracyassessment only ifreference materialsprepared from actualcontaminated fish orshellfish are not available.

Method validation: As many as requiredto assess accuracy (and precision) ofmethod before routine analysis ofsamples (i.e., when using a method forthe first time or after any methodmodification).Routine accuracy assessment. One per20 samples or one per batch, whicheveris more frequent.

Determined by program manager.Organics: %R = 70-130.d

Metals: %R = 85-115.d

NA

Determine cause of problem (e.g.,inaccurate calibration, inaccuratepreparation of control samples),take appropriate corrective action,and reanalyze all suspect samplesor flag all suspect data. Zeropercent recovery requires rejectionof all suspect data.
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-19

Table 8-6. (continued)

Sample type

(definition;

specifications) Objective

Recommended

frequency of analysisa

Recommended

control limitsb

Recommended 

corrective action

(continued)

Matrix spikes(composite tissue homogenatesof field samples to which knownamounts of target analytes havebeen added; 0.5 to 5 times theconcentration of the analyte ofinterest or 5 times the MQL)

Assess matrix effects andaccuracy (%R) routinely.

One per 20 samples or one per batch,whichever is more frequent.

Organics: %R $50 with good

 precision.Metals: %R = 75-125.

Determine cause of problem (e.g.,incomplete extraction or digestion,contamination), take appropriatecorrective action, and reanalyze allsuspect samples or flag all suspectdata. Zero percent recoveryrequires rejection of all suspectdata.

Matrix spike replicates(replicate aliquots of matrix spikesamples; 0.5 to 5 times theconcentration of the analyte ofinterest or 5 times the MQL)

Assess method precisionroutinely.

One duplicate per 20 samples or one perbatch, whichever is more frequent.

Organics: A difference of no

more than a factor of 2 amongreplicates (i.e., approximately 50%coefficient of variation). Note: Pooling of variances induplicate analyses from differentsample batches is recommendedfor estimating the standarddeviation or coefficient of variationof replicate analyses. Metals: |RPD| #20 for duplicates.

Determine cause of problem (e.g.,incomplete extraction or digestion,contamination, instrument instabilityor malfunction), take appropriatecorrective action, and reanalyze allsuspect samples or flag all suspectdata.

Laboratory replicatese

(replicate aliquots of compositetissue homogenates of fieldsamples)

Assess method precisionroutinely.

One blind duplicate sample per 20samples or one per batch, whichever ismore frequent.

Organics: A difference of no

more than a factor of 2 amongreplicates (i.e., approximately 50%coefficient of variation). Note: Pooling of variances induplicate analyses from differentsample batches is recommendedfor estimating the standarddeviation or coefficient of variationof replicate analyses.

Metals: |RPD| #20 for duplicates.

Determine cause of problem (e.g.,composite sample nothomogeneous, instrumentinstability or malfunction), takeappropriate corrective action, andreanalyze all suspect samples orflag all suspect data.

Analytical Replicates(replicate aliquots of final sampleextract or digestate)

Assess analyticalprecision.

Duplicate injections for all metalanalyses.f Determined by program manager.g Determine cause of problem (e.g., instrument instability or

malfunction), take appropriatecorrective action, and reanalyzesample.
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-20

Table 8-6. (continued)

Sample type

(definition;

specifications) Objective

Recommended

frequency of analysisa

Recommended

control limitsb

Recommended 

corrective action

(continued)

Field replicates(replicate composite tissuesamples)

Assess total variability(i.e., population variability,field or samplingvariability, and analyticalmethod variability).

Screening studies: OPTIONAL; ifprogram resources allow, a minimum ofone blind replicate (i.e., duplicate) foreach primary target species at 10 percentof screening sites.g

Intensive studies: Blind replicatesamples for each target species (andsize, age or sex class, if appropriate) ateach sampling site. Number of replicatesdetermined by program manager (seeSection 6.1.2.7).

Determined by program manager.g

Determined by program manager.g

Determined by program manager.Determined by program manager.

Contamination AssessmentBlanks (field, method,processing, bottle,reagent)(see Section 8.3.3.6 fordefinitions)

Assess contaminationfrom equipment, reagents,etc.

One field blank per sampling site.One method blank per 20 samples or oneper batch, whichever is more frequent. Atleast one processing blank per study. Atleast one bottle blank per lot or per batchof samples, whichever is more frequent. One reagent blank prior to use of a newbatch of reagent and whenever methodblank exceeds control limits.

Concentration of any analyte <MDL orMQL, as determined by programmanager.

Determine cause of problem (e.g.,contaminated reagents,equipment), remove sources ofcontamination, and reanalyze allsuspect samples or flag all suspectdata.

Routine Monitoring of Method Performance for Organic AnalysesSurrogate spikes (see Section8.3.3.7.1 for definition)

Prepared from isotopicallylabeled target analytes

Assess method performance and estimaterecovery of organic targetanalytes analyzed byGC/MS. Determine RRFsof organic target analytesquantitated by isotopedilution techniques. 

In every calibration standard, sample,and blank analyzed for organics byisotope dilution GC/MS; added tosamples prior to extraction.

Determined by program manager. Determine cause of problem (e.g.,

incomplete extraction or digestion,contamination, inaccuratepreparation of internal standard),take appropriate corrective action,and reanalyze all suspect samplesor flag all suspect data.
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-21

Table 8-6. (continued)

Sample type

(definition;

specifications) Objective

Recommended

frequency of analysisa

Recommended

control limitsb

Recommended 

corrective action

(continued)

Prepared from othersurrogate compounds

Assess methodperformance and estimatethe recovery of organictarget analytes analyzedby GC/MS or GC/ECD.

In every calibration standard, sample,and blank analyzed for organics, unlessisotope dilution technique is used:Semivolatiles:

3 for neutral fraction2 for acid fraction

Volatiles: 3Pesticides/PCBs: 1Added to samples prior to extraction.

Determined by program manageraccording to most recent EPA CLPguidelines.h

Determine cause of problem (e.g.,incomplete extraction or digestion,contamination, inaccuratepreparation of surrogates), takeappropriate corrective action, andreanalyze all suspect samples orflag all suspect data.

External QA AssessmentAccuracy-based performanceevaluation samples(QA samples from NOAAinterlaboratory comparisonprogram; see Section 8.3.3.8.1)

Initial demonstration oflaboratory capability.Ongoing demonstration oflaboratory capability.

Once prior to routine analysis of fieldsamples (blind).One exercise (four to six samples) peryear (blind).

Organics: %R=70-130.d

Metals: %R=85-115.dDetermined by NOAA. Based onconsensus value of all participatinglaboratories.

Determine cause of problem andreanalyze sample. Do not beginanalysis of field samples untilperformance evaluation sampleresults are acceptable.Determine cause of problem. Donot continue analysis of fieldsamples until laboratory capabilityis clearly demonstrated.
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-22

Table 8-6. (continued)

Sample type

(definition;

specifications) Objective

Recommended

frequency of analysisa

Recommended

control limitsb

Recommended 

corrective action

(continued)

Split samples(laboratory replicates analyzedby different laboratories; seeSection 8.3.3.8.2)

Assess interlaboratorycomparability.

5-10% of composite homogenates splitbetween states and/or Regions thatroutinely share monitoring results, or asdetermined by program managers.g

Determined by program managers. Review sampling and analytical

methods. Identify sources ofnoncomparability. Standardize andvalidate methods to documentcomparability.

CLP = Contract laboratory program.CRM = Certified reference material (see Section

8.3.3.1).

GC/ECD = Gas chromatography/electron capture

detection.

GC/MS = Gas chromatography/mass spectrometry.MDL = Method detection limit (see Section

8.3.3.3.1).

MQL = Method quantitation limit (see Section

8.3.3.3.2).

NA = Not applicable.NOAA = National Oceanic and Atmospheric

Administration.

PCBs = Polychlorinated biphenyls.QA = Quality assurance.%R = Percent recovery (see Sections 8.3.3.4 and

8.3.3.7.1).

RF = Response factor (see Section 8.3.3.2.1).

RPD = Relative percent difference (see Section

8.3.3.5).

RRF = Relative response factor (see Section

8.3.3.2.1).

RSD = Relative standard deviation (see Section

8.3.3.5).

SRM = Standard reference material (see Section

8.3.3.1).

a Recommended frequencies are based primarily on recommendations in U.S. EPA (1986d, 1987e, 1989c, 1991b, 1991c), Puget Sound Estuary Program (1990d, 1990e), and

Battelle (1989b). b From Puget Sound Estuary Program (1990d, 1990e) action limits, except where otherwise noted. Note: Individual programs may require more stringent control limits. It is theresponsibility of each program manager to set control limits that will ensure that the measurement data meet program data quality objectives. c As available (see Table 8-8 and Appendix M). d From U.S.EPA (1991e). e Sometimes referred to as analytical replicates (e.g., in Puget Sound Estuary Program, 1990d). f From U.S. EPA (1987e). g Recommended by EPA for this guidance document. h From U.S. EPA (1991b, 1991c).
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

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Table 8-7. Minimum Recommended QA and QC Samples for

Routine Analysis of Target Analytesa

Sample Type

Target analyte

Metals Organics

Accuracy-based performance

evaluation sampleb

Once prior to routine

analysis of field samples,

plus one exercise (four

to six samples) per year.

Once prior to routine

analysis of field samples,

plus one exercise (four to

six samples) per year.

Method blank 1 1 

Laboratory duplicate 1 1 

Matrix spike/matrix spike replicate 1 1 

Laboratory control sample

(SRM or CRM, if available)

1 1 

Calibration check standard 2c 2c

Surrogate spike (isotopically labeled target

analyte or other surrogate compound added

prior to extraction)

NA Each sample

Instrument (injection) internal standard;

added prior to injection

NA Each calibration or

calibration check standard

and each sample or blank

analyzed by GC/MSd

CRM = Certified reference material (see Section

8.3.3.1).

GC/MS = Gas chromatography/mass spectroscopy.

NA = Not applicable.

QA = Quality assurance.

QC = Quality control.

SRM = Standard reference material (see Section

8.3.3.1).

a Unless otherwise specified, the number given is the recommended number of QC samples per 20 samples or

per batch, whichever is more frequent. Additional method-specific QC requirements should always be

followed provided these minimum requirements have been met.

b QA samples from National Oceanic and Atmospheric Administration interlaboratory comparison program (see

Section 8.3.3.8.1).

c One every 10 samples (plus one at beginning and end of each analytical run).

d Optional for analyses by GC/electron capture detection (ECD), GC/flame ionization detection (FID), or GC

with other nonspecific detectors.
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

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8.3.3.1 Reference Materials—

The appropriate use of reference materials is an essential part of good QA and

QC practices for analytical chemistry. The following definitions of reference

materials (Puget Sound Estuary Program, 1990d) are used in this guidance

document:

• A reference material is any material or substance of which one or more

properties have been sufficiently well established to allow its use for instrument

calibration, method evaluation, or characterization of other materials.

• A certified reference material (CRM) is a reference material of which the

value(s) of one or more properties has (have) been certified by a variety of

technically valid procedures. CRMs are accompanied by or traceable to a

certificate or other documentation that is issued by the certifying organization

(e.g., U.S. EPA, NIST, National Research Council of Canada [NRCC]). 

• A standard reference material (SRM) is a CRM issued by the NIST.

Reference materials may be used to (1) provide information on method accuracy

and, when analyzed in replicate, on precision, and (2) obtain estimates of

intermethod and/or interlaboratory comparability. An excellent discussion of the

use of reference materials in QA and QC procedures is given in Taylor (1985).

The following general guidelines should be followed to ensure proper use of

reference materials (NOAA, 1992):

• When used to assess the accuracy of an analytical method, the matrix of the

reference material should be as similar as possible to that of the samples of

interest. If reference materials in matrices other than fish or shellfish tissue

are used, possible matrix effects should be addressed in the final data

analysis or interpretation.

• Concentrations of reference materials should cover the range of possible

concentrations in the samples of interest. Note: Because of a lack of lowand high-concentration reference materials for most analytes in fish and

shellfish tissue matrices, potential problems at low or high concentrations

often cannot be documented.

• Reference materials should be analyzed prior to beginning the analyses of

field samples to assess laboratory capability and regularly thereafter to detect

and document any changes in laboratory performance over time. Appropriate

corrective action should be taken whenever changes are observed outside

specified performance limits (e.g., accuracy, precision).

• If possible, reference material samples should be introduced into the sample

stream as double blinds, that is, with identity and concentration unknown to

the analyst. However, because of the limited number of certified fish and
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

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shellfish tissue reference materials available, the results of analyses of these

materials may be biased by an analyst’s increasing ability to recognize these

materials with increased use. 

• Results of reference material analyses are essential to assess interlaboratory

or intermethod comparability. However, the results of sample analyses should

not be corrected based on percent recoveries of reference materials. Final

reported results should include both uncorrected sample results and percent

recoveries of reference materials.

Sources of reference materials for the analysis of priority pollutants and selected

related compounds in fish and shellfish tissues are given in Appendix M.

Available marine or estuarine tissue reference materials that may be appropriate

for use by analytical laboratories in fish and shellfish contaminant monitoring

programs are given in Table 8-8.

8.3.3.2 Calibration and Calibration Checks—

General guidelines for initial calibration and routine calibration checks are

provided in this section. Method-specific calibration procedures are included in

the references in Table 8-2. It is the responsibility of each program manager to

ensure that proper calibration procedures are developed and followed for each

analytical method to ensure the accuracy of the measurement data. 

All analytical instruments and equipment should be maintained and calibrated

properly to ensure optimum operating conditions throughout a measurement

program. Calibration and maintenance procedures should be performed

according to SOPs based on the manufacturers’ specifications and the

requirements of specific analytical procedures. Calibration procedures must

include provisions for documenting calibration frequencies, conditions, standards,

and results to describe adequately the calibration history of each measurement

system. Calibration records should be inspected regularly to ensure that these

procedures are being performed at the required frequency and according to

established SOPs. Any deficiencies in the records or deviations from established

procedures should be documented and appropriate corrective action taken.

Calibration standards of known and documented accuracy must be used to

ensure the accuracy of the analytical data. Each laboratory should have a

program for verifying the accuracy and traceability of calibration standards against

the highest quality standards available. If possible, NIST-SRMs or other certified

reference standards should be used for calibration standards (see Section 8.3.3.4

and Appendix M). A log of all calibration materials and standard solutions should

be maintained. Appropriate storage conditions (i.e., container specifications,

shelf-life, temperature, humidity, light condition) should be documented and

maintained.
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

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Table 8-8. Fish and Shellfish Tissue Reference Materials

Identification

code Analytes Source Matrix

DOLT-1 Elements NRCC Dogfish liver (freeze-dried)

DORM-1 Elements NRCC Dogfish muscle (freeze-dried)

LUTS-1 Elements NRCC Non-defatted lobster hepatopancreas

TORT-1 Elements NRCC Lobster hepatopancreas

GBW-08571 Elements NRCCRM Mussel tissue (freeze-dried)

GBW-08572 Elements NRCCRM Prawn tissue

MA-A-1/OC Organic compounds IAEA Copepod homogenate (freeze-dried)

MA-A-3/OC Organic compounds IAEA Shrimp homogenate (freeze-dried)

MA-B-3/OC Organic compounds IAEA Fish tissue (freeze-dried)

MA-M-2/OC Organic compounds IAEA Mussel tissue

MA-A-1/TM Elements IAEA Copepod homogenate (freeze-dried)

MA-A-2/TM Elements IAEA Fish flesh homogenate

MA-B-3/TM Elements IAEA Fish tissue (freeze-dried)

MA-B-3/RN Isotopes IAEA Fish tissue (freeze-dried)

IAEA-350 Elements IAEA Tuna homogenate (freeze-dried)

IAEA-351 Organic compounds IAEA Tuna homogenate (freeze-dried)

IAEA-352 Isotopes IAEA Tuna homogenate (freeze-dried)

CRM-278 Elements BCR Mussel tissue (freeze-dried)

CRM-422 Elements BCR Cod muscle (freeze-dried)

EPA-FISH Pesticides EPA1 Fish tissue

EPA-SRS903 Chlordane EPA2 Fish tissue

EPA-0952 Mercury EPA1 Fish tissue

EPA-2165 Mercury EPA1 Fish tissue

RM-50 Elements NIST Albacore tuna (freeze-dried)

SRM-1566a Elements NIST Oyster tissue (freeze-dried)

SRM-1974 Organic compounds NIST Mussel tissue (frozen)

SRM-2974 Organic compounds NIST Mussel tissue (freeze-dried)

NIES-6 Elements NIES Mussel tissue

Sources:

BCR = Community Bureau of Reference, Commission of the European Communities, Directorate General for

Science, Research and Development, 200 rue de la Loi, B-1049 Brussels, Belgium.

EPA = U.S. Environmental Protection Agency, Quality Assurance Branch, EMSL-Cincinnati, Cincinnati, OH,

45268, USA. (EPA1: Material available from Supelco, Inc., Supelco Park, Bellefonte, PA, 16823-

0048, USA. EPA2: Material available from Fisher Scientific, 711 Forbes Ave., Pittsburgh, PA 15219.)

IAEA = International Atomic Energy Agency, Analytical Quality Control Service, Laboratory Seibersdorf, P. O.

Box 100, A-1400 Vienna, Austria.

NRCCRM = National Research Center for CRMs, Office of CRMs, No. 7, District 11, Hepingjie, Chaoyangqu,

Beijing, 100013, China.

NRCC = National Research Council of Canada, Institute for Environmental Chemistry, Marine Analytical

Chemistry Standards Program, Division of Chemistry, Montreal Road, Ottawa, Ontario K1A 0R9,

Canada.

NIST = National Institute of Standards and Technology, Office of Standard Reference Materials, Gaithersburg,

MD, 20899, USA.

NIES = National Institute for Environmental Studies, Yatabe-machi, Tsukuba, Ibaraki, 305, Japan.
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

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8.3.3.2.1 Initial and routine calibration

Prior to beginning routine analyses of samples, a minimum of three (and

preferably five) calibration standards should be used to construct a calibration

curve for each target analyte, covering the normal working range of the instrument

or the expected target analyte concentration range of the samples to be analyzed.

The lowest-concentration calibration standard should be at or near the estimated

method detection limit (see Section 8.3.3.3.1). Calibration standards should be

prepared in the same matrix (i.e., solvent) as the final sample extract or digestate.

Criteria for acceptable calibration (e.g., acceptable limits for r2

, slope, intercept,

percent recovery, response factors) should be established for each analytical

method. If these control limits are exceeded, the source of the problem (e.g.,

inaccurate standards, instrument instability or malfunction) should be identified

and appropriate corrective action taken. No analyses should be performed until

acceptable calibration has been achieved and documented.

In addition to the initial calibration, an established schedule for the routine

calibration and maintenance of analytical instruments should be followed, based

on manufacturers’ specifications, historical data, and specific procedural requirements. At a minimum, calibration should be performed each time an instrument

is set up for analysis, after any major disruption or failure, after any major

maintenance, and whenever a calibration check exceeds the recommended

control limits (see Table 8-6).

Two types of calibration procedures are used in the analytical methods recommended for the quantitation of target analytes: external calibration and internal

standard calibration.

External calibration

In external calibration, calibration standards with known concentrations of target

analytes are analyzed, independent of samples, to establish the relationship

between instrument response and target analyte concentration. External

calibration is used for the analyses of metals and, at the option of the program

manager, for the analyses of organics by gas chromatography/electron capture

detection (GC/ECD), gas chromatography/flame ionization detection (GC/FID),

or GC methods using other nonspecific detectors.

External calibration for metals analysis is considered acceptable if the percent

recovery of all calibration standards is between 95 and 105 percent; external

calibration for organic analyses is considered acceptable if the relative standard

deviation (RSD) of the response factors (RFs) is 20 percent (see Table 8-6). If

these limits are exceeded, the initial calibration should be repeated.
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

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Internal standard calibration

Calibration of GC/mass spectrometry (MS) systems used for the analysis of

organic target analytes requires the addition of an internal standard to each

calibration standard and determination of the response of the target analyte of

interest relative to that of the internal standard. Internal standard calibration may

also be used with nonspecific detector GC methods such as GC/ECD and

GC/FID. Internal standards used to determine the relative response factors

(RRFs) are termed instrument or injection internal standards (Puget Sound

Estuary Program, 1990d; U.S. EPA, 1991e). The addition of instrument internal

standards to both calibration standards and sample extracts ensures rigorous

quantitation, particularly accounting for shifts in retention times of target analytes

in complex sample extracts relative to calibration standards. Recommended

instrument internal standards for semivolatile organic compounds are included in

analytical methods for these compounds (see references in Table 8-2).

The RRF for each target analyte is calculated for each calibration standard as

follows:

RRFt

 = (At) (Cis) / (Ais) (Ct) (8-1)

where

At = Measured response (integrated peak area) for the target analyte

Cis = Concentration of the instrument internal standard in the calibration

standard

Ais = Measured response (integrated peak area) for the instrument internal

standard

Ct = Concentration of the target analyte in the calibration standard.

If the RSD of the average RRFt

 for all calibration standards (RRFt

———— ) is 30 percent,

RRFt

 can be assumed to be constant across the working calibration range and

RRFt

———— can be used to quantitate target analyte concentrations in the samples as

follows:

Ct

 (ppm or ppb, wet weight) = (At) (Cis) (Ve) / (Ais) (RRFt

———— ) (W) (8-2)

where

Ct = Concentration of the target analyte in the sample

Cis = Concentration of the instrument internal standard in the sample extract

Ve = Volume of the final sample extract (mL)

W = Weight of sample extracted (g)
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

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and At

, Ais, and RRFt

———— are defined as in Equation (8-1).

If the RSD of RRFt

———— for all calibration standards is >30 percent, the initial

calibration should be repeated (see Table 8-6).

8.3.3.2.2 Routine calibration checks

After initial calibration has been achieved and prior to the routine analyses of

samples, the accuracy of the calibration should be verified by the analysis of a

calibration check standard. A calibration check standard is a mid-range

calibration standard that has been prepared independently (i.e., using a different

stock) from the initial calibration standards. When internal standard calibration is

being used, an instrument internal standard must be added to each calibration

check standard.

Routine calibration checks should be conducted often enough throughout each

analysis run to ensure adequate maintenance of instrument calibration (see

Table 8-6). A calibration check should always be performed after analyzing the

last sample in a batch and at the end of each analysis run.

If a calibration check does not fall within specified calibration control limits, the

source of the problem should be determined and appropriate corrective action

taken (see Table 8-6). After acceptable calibration has been reestablished, all

suspect analyses should be repeated. If resources permit, it is recommended that

all samples after the last acceptable calibration check be reanalyzed. Otherwise,

the last sample analyzed before the unacceptable calibration check should be

reanalyzed first and reanalysis of samples should continue in reverse order until

the difference between the reanalysis and initial results is within the control limits

specified in Table 8-6. If reanalysis is not possible, all suspect data (i.e., since the

last acceptable calibration check) should be identified clearly in the laboratory

records and the data report. 

8.3.3.2.3 Calibration range and data reporting

As noted in Section 8.3.2.1, the lowest-concentration calibration standard should

be at or near the method detection limit. The highest-concentration calibration

standard should be selected to cover the full range of expected concentrations of

the target analyte in fish and shellfish tissue samples. If a sample concentration

occurs outside the calibration range, the sample should be diluted or concentrated

as appropriate and reanalyzed or the calibration range should be extended.

Extremely high concentrations of organic compounds may indicate that the

extraction capabilities of the method have been saturated and extraction of a

smaller sample or modification of the extraction procedure may be required.

All reported concentrations must be within the upper limit of the demonstrated

working calibration range. Procedures for reporting data, with appropriate
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

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qualifications for data below method detection and quantitation limits, are given

in Section 8.3.3.3.3.

8.3.3.3 Assessment of Detection and Quantitation Limits—

It is the responsibility of each laboratory to determine appropriate detection and

quantitation limits for each analytical method for each target analyte in a fish or

shellfish tissue matrix. When available scientific literature demonstrates that the

selected SVs are analytically attainable, the laboratory is responsible for ensuring

that these limits are sufficiently low to allow reliable quantitation of the analyte at

or below the selected SVs (see Section 5.2). Detection and quantitation limits

must be determined prior to the use of any method for routine analyses and after

any significant changes are made to a method during routine analyses. Several

factors influence achievable detection and quantitation limits regardless of the

specific analytical procedure. These include amount of sample available, matrix

interferences, and stability of the instrumentation. The limits of detection given

in Table 8-4 are considered to be representative of typically attainable values.

Depending upon individual laboratory capabilities and fish tissue matrix properties,

it should be noted that SVs for some recommended target analytes (e.g.,

inorganic arsenic, dieldrin, heptachlor epoxide, toxaphene, PCBs, and dioxins/

furans) may not always be analytically attainable quantitation limits. In these

instances, all historic and current data on contaminant sources and on water,

sediment, and fish and shellfish contaminant tissue data should be reviewed to

provide additional information that could aid in the risk assessment process and

in making risk management decisions.

The EPA has previously issued guidance on detection limits for trace metal and

organic compounds for analytical methods used in chemical contaminant

monitoring programs (U.S. EPA, 1985a). However, at present there is no clear

consensus among analytical chemists on a standard procedure for determining

and reporting the limits of detection and quantitation of analytical procedures.

Furthermore, detection and quantitation limits reported in the literature are seldom

clearly defined. Reported detection limits may be based on instrument sensitivity

or determined from the analyses of method blanks or low-level matrix spikes;

quantitation limits may be determined from the analyses of method blanks or

low-level matrix spikes (Puget Sound Estuary Program, 1990d).

8.3.3.3.1 Detection limits

The EPA recommends that the method detection limit (MDL) defined below and

determined according to 40 CFR 136, Appendix B, be used to establish the limits

of detection for the analytical methods used for analyses of all target analytes:

• Method Detection Limit: The minimum concentration of an analyte in a

given matrix (i.e., fish or shellfish tissue homogenates for the purposes of this

guidance) that can be measured and reported with 99 percent confidence that

the concentration is greater than zero. The MDL is determined by multiplying
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

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the appropriate (i.e., n-1 degrees of freedom) one-sided 99 percent Student’s

t-statistic (t0.99) by the standard deviation (S) obtained from a minimum of

seven replicate analyses of a spiked matrix sample containing the analyte

of interest at a concentration three to five times the estimated MDL (Glaser et

al., 1981; 40 CFR 136, Appendix B):

MDL = (t0.99) (S). (8-3)

It is important to emphasize that all sample processing steps of the analytical

method (e.g., digestion, extraction, cleanup) must be included in the

determination of the MDL.

In addition to the MDL, three other types of detection limits have been defined by

the American Chemical Society Committee on Environmental Improvement (Keith,

1991a):

• Instrument Detection Limit (IDL): The smallest signal above background

noise that an instrument can detect reliably.

• Limit of Detection (LOD): The lowest concentration that can be determined

to be statistically different from a method blank at a specified level of

confidence. The recommended value for the LOD is three times the standard

deviation of the blank in replicate analyses, corresponding to a 99 percent

confidence level.

• Reliable Detection Limit (RDL): The concentration level of an analyte in a

given matrix at which a detection decision is extremely likely. The RDL is

generally set higher than the MDL. When RDL=MDL, the risk of a false

positive at 3 from zero is <1 percent, whereas the corresponding risk of a

false negative is 50 percent. When RDL=2MDL, the risk of either a false

positive or a false negative at 3 from zero is <1 percent.

Each of these estimates has its practical limitations. The IDL does not account

for possible blank contaminants or matrix interferences. The LOD accounts for

blank contaminants but not for matrix effects or interferences. In some instances,

the relatively high value of the MDL or RDL may be too stringent and result in the

rejection of valid data; however, these are the only detection limit estimates that

account for matrix effects and interferences and provide a high level of statistical

confidence in sample results. The MDL is the recommended detection limit in the

EPA EMAP-NC Program (U.S. EPA, 1991e).

The MDL, expressed as the concentration of target analyte in fish tissue, is

calculated from the measured MDL of the target analyte in the sample extract or

digestate according to the following equation:

MDLtissue (ppm or ppb) = (MDLextract V) /W (8-4)
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

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where

V = Final extract or digestate volume, after dilution or concentration (mL)

W = Weight of sample digested or extracted (g).

Equation 8-4 clearly illustrates that the MDL in tissue may be improved (reduced)

by increasing the sample weight (W) and/or decreasing the final extract or

digestate volume (V).

The initial MDL is a statistically derived empirical value that may differ in actual

samples depending on several factors, including sample size, matrix effects, and

percent moisture. Therefore, it is recommended that each laboratory reevaluate

annually all MDLs for the analytical methods used for the sample matrices

typically encountered (U.S. EPA, 1991e).

Experienced analysts may use their best professional judgment to adjust the

measured MDL to a lower "typically achievable" detection limit (Puget Sound

Estuary Program, 1990e; U.S. EPA, 1985a) or to derive other estimates of

detection limits. For example, EPA recommends the use of lower limits of

detection (LLDs) for GG/MS methods used to analyze organic pollutants in

bioaccumulation monitoring programs (U.S. EPA, 1986a). Estimation of the LLD

for a given analyte involves determining the noise level in the retention window for

the quantitation mass of the analyte for at least three field samples in the sample

set being analyzed. The LLD is then estimated as the concentration

corresponding to the signal required to exceed the average noise level observed

by at least a factor of 2. Based on the best professional judgment of the analyst,

this LLD is applied to samples in the set with comparable or lower interference;

samples with significantly higher interferences (i.e., by at least a factor of 2) are

assigned correspondingly higher LLDs. LLDs are greater than IDLs but usually

are less than the more rigorously defined MDLs. Thus, data quantified between

the LLD and the MDL have a lower statistical confidence associated with them

than data quantified above the MDL. However, these data are considered valid

and useful in assessing low-level environmental contamination. 

If estimates of detection limits other than the MDL are developed and used to

qualify reported data, they should be clearly defined in the analytical SOPs and

in all data reports, and their relationship to the MDL should be clearly described.

8.3.3.3.2 Quantitation limits

In addition to the MDL, a method quantitation limit (MQL), or minimum concentration allowed to be reported at a specified level of confidence without qualifications,

should be derived for each analyte. Ideally, MQLs should account for matrix

effects and interferences. The MQL can be greater than or equal to the MDL. At

present, there is no consistent guidance in the scientific literature for determining

MQLs; therefore, it is not possible to provide specific recommendations for

determining these limits at this time.
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

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The American Chemical Society Committee on Environmental Improvement

(Keith, 1991b; Keith et al., 1983) has defined one type of quantitation limit:

• Limit of Quantitation (LOQ): The concentration above which quantitative

results may be obtained with a specified degree of confidence. The

recommended value for the LOQ is 10 times the standard deviation of a

method blank in replicate analyses, corresponding to an uncertainty of ±30

percent in the measured value (10 ± 3) at the 99 percent confidence level.

The LOQ is the recommended quantitation limit in the EPA EMAP-NC Program

(U.S. EPA, 1991e). However, the LOQ does not account for matrix effects or

interferences.

The U.S. EPA (1986d) has defined another type of quantitation limit:

• Practical Quantitation Limit (PQL): The lowest concentration that can be

reliably reported within specified limits of precision and accuracy under routine

laboratory operating conditions.

The Puget Sound Estuary Program (1990d) and the National Dioxin Study (U.S.

EPA, 1987d) used a PQL based on the lowest concentration of the initial

calibration curve (C, in μg/mL), the amount of sample typically analyzed (W, in g),

and the final extract volume (V, in mL) of that method: 

PQL g g ppm (8-5) C g mL V mL

W g ( / [ ]) (/ ) ()( ) m m = ·

However, this PQL is also applicable only to samples without substantial matrix

effects or interferences.

A reliable detection limit (RDL) equal to 2 MDL may also be used as an estimate

of the MQL (see Section 8.3.3.3.1). The RDL accounts for matrix effects and

provides a high level of statistical confidence in analytical results.

Analysts must use their expertise and professional judgment to determine the best

estimate of the MQL for each target analyte. MQLs, including the estimated

degree of confidence in analyte concentrations above the quantitation limit, should

be clearly defined in the analytical SOPs and in all data reports.

8.3.3.3.3 Use of detection and quantitation limits in reporting data

The analytical laboratory does not have responsibility or authority to censor data.

Therefore, all data should be reported with complete documentation of limitations

and problems. Method detection and quantitation limits should be used to qualify

reported data for each composite sample as follows (Keith, 1991b):
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-34

• "Zero" concentration (no observed response) should be reported as not

detected (ND) with the MDL noted, e.g., "ND(MDL=X)".

• Concentrations below the MDL should be reported with the qualification that

they are below the MDL.

• Concentrations between the MDL and the MQL should be reported with the

qualification that they are below the quantitation limit.

• Concentrations at or above the MQL may be reported and used without

qualification.

The use of laboratory data for comparing target analyte concentrations to SVs in

screening and intensive studies is discussed in Sections 9.1.1 and 9.1.2.

8.3.3.4 Assessment of Method Accuracy—

The accuracy of each analytical method should be assessed and documented for

each target analyte of interest, in a fish or shellfish tissue matrix, prior to

beginning routine analyses and on a regular basis during routine analyses.

Method accuracy may be assessed by analysis of appropriate reference materials

(i.e., SRMs or CRMs prepared from actual contaminated fish or shellfish tissue,

see Table 8-8, laboratory control samples (i.e., accuracy-based samples

consisting of fish and shellfish tissue homogenates spiked with compounds

representative of the target analytes of interest), and/or matrix spikes. If

possible, laboratory control samples should be SRMs or CRMs. Note: Only the

analysis of fish or shellfish tissue SRMs or CRMs prepared from actual

contaminated fish or shellfish tissue allows rigorous assessment of total method

accuracy, including the accuracy with which an extraction or digestion procedure

isolates the target analyte of interest from actual contaminated fish or shellfish.

The analysis of spiked laboratory control samples or matrix spikes provides an

assessment of method accuracy including sample handling and analysis

procedures but does not allow rigorous assessment of the accuracy or efficiency

of extraction or digestion procedures for actual contaminated fish or shellfish.

Consequently, these samples should not be used for the primary assessment of

total method accuracy unless SRMs or CRMs prepared from actual contaminated

fish or shellfish tissue are not available.

The concentrations of target analytes in samples used to assess accuracy should

fall within the range of concentrations found in the field samples; however, this

may not always be possible for reference materials or laboratory control samples

because of the limited number of these samples available in fish and shellfish

tissue matrices (see Table 8-8). Matrix spike samples should be prepared using

spike concentrations approximately equal to the concentrations found in the

unspiked samples. An acceptable range of spike concentrations is 0.5 to 5 times
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-35

the expected sample concentrations (U.S. EPA, 1987e). Spikes should always

be added to the sample homogenates prior to digestion or extraction.

Accuracy is calculated as percent recovery from the analysis of reference

materials, or laboratory control samples, as follows:

% Recovery = 100 (M/T) (8-6)

where

M = Measured value of the concentration of target analyte

T = "True" value of the concentration of target analyte.

Accuracy is calculated as percent recovery from the analysis of matrix spike

samples as follows:

% Recovery = [(Ms - Mu)/Ts] x 100 (8-7)

where

Ms = Measured concentration of target analyte in the spiked sample

Mu = Measured concentration of target analyte in the unspiked sample

Ts = "True" concentration of target analyte added to the spiked sample.

When sample concentrations are less than the MDL, the value of one-half the

MDL should be used as the concentration of the unspiked sample (Mu) in

calculating spike recoveries.

8.3.3.4.1 Initial assessment of method accuracy

As discussed above, method accuracy should be assessed initially by analyzing

appropriate SRMs or CRMs that are prepared from actual contaminated fish or

shellfish tissue. The number of reference samples required to be analyzed for the

initial assessment of method accuracy should be determined by each laboratory

for each analytical procedure with concurrence of the program manager. If such

SRMs or CRMs are not available, laboratory control samples or matrix spikes may

be used for initial assessment of method accuracy.

8.3.3.4.2 Routine assessment of method accuracy

Laboratory control samples and matrix spikes should be analyzed for continuous

assessment of accuracy during routine analyses. It is recommended that one

laboratory control sample and one matrix spike sample be analyzed with every 20

samples or with each sample batch, whichever is more frequent (Puget Sound

Estuary Program, 1990d, 1990e). Ideally, CRMs or SRMs should also be

analyzed at this recommended frequency; however, limited availability and cost

of these materials may make this impractical.
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-36

For organic compounds, isotopically labeled or surrogate recovery standards that

must be added to each sample to monitor overall method performance also

provide an assessment of method accuracy (see Section 8.3.3.7.1).

Percent recovery values for spiked samples must fall within established control

limits (see Table 8-6). If the percent recovery falls outside the control limit, the

analyses should be discontinued, appropriate corrective action taken, and, if

possible, the samples associated with the spike reanalyzed. If reanalysis is not

possible, all suspect data should be clearly identified.

Note: Reported data should not be corrected for percent recoveries. Recovery

data should be reported for each sample to facilitate proper evaluation and use

of analytical results.

Poor performance on the analysis of reference materials or poor spike recovery

may be caused by inadequate mixing of the composite homogenate sample

before aliquotting, inconsistent digestion or extraction procedures, matrix

interferences, or instrumentation problems. If replicate analyses are acceptable

(see Section 8.3.3.5), matrix interferences or loss of target analytes during sample

preparation are indicated. To check for loss of target analytes during sample

preparation, a step-by-step examination of the procedure using spiked blanks

should be conducted. For example, to check for loss of metal target analytes

during digestion, a postdigestion spike should be prepared and analyzed and the

results compared with those from a predigestion spike. If the results are

significantly different, the digestion technique should be modified to obtain

acceptable recoveries. If there is no significant difference in the results of preand postdigestion spikes, the sample should be diluted by at least a factor of 5

and reanalyzed. If spike recovery is still poor, then the method of standard

additions or use of a matrix modifier is indicated (U.S. EPA, 1987e).

8.3.3.5 Assessment of Method Precision—

The precision of each analytical method should be assessed and documented for

each target analyte prior to the performance of routine analyses and on a regular

basis during routine analysis.

Precision is defined as the agreement among a set of replicate measurements

without assumption of knowledge of the true value. Method precision (i.e., total

variability due to sample preparation and analysis) is estimated by means of the

analyses of duplicate or replicate tissue homogenate samples containing

concentrations of the target analyte of interest above the MDL. All samples used

for assessment of total method precision must be carried through the complete

analytical procedure, including extraction or digestion.

The most commonly used estimates of precision are the relative standard deviation or coefficient of variation (CV) for multiple samples, and the relative percent
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-37

difference (RPD) when only two samples are available. These are defined as

follows:

RSD = CV = 100 S/x ̄i (8-8)

where

S = Standard deviation of the xi

 measurements

x ̄i = Arithmetic mean of the xi

 measurements

and

RPD = 100 {(x1 - x2)/[(x1 + x2)/2]} . (8-9)

8.3.3.5.1 Initial assessment of method precision

Method precision should be assessed prior to routine sample analyses by

analyzing replicate samples of the same reference materials, laboratory control

samples, and/or matrix spikes that are used for initial assessment of method

accuracy (see Section 8.3.3.4.1). The number of replicates required to be

analyzed for the initial assessment of method precision should be determined by

each laboratory for each analytical procedure with concurrence of the program

manager. Because precision may be concentration-dependent, initial assessments of precision across the estimated working range should be obtained.

8.3.3.5.2 Routine assessment of method precision

Ongoing assessment of method precision during routine analysis should be

performed by analyzing replicate aliquots of tissue homogenate samples taken

prior to sample extraction or digestion (i.e., laboratory replicates) and matrix

spike replicates. Matrix spike concentrations should approximate unspiked

sample concentrations; an acceptable range for spike concentrations is 0.5 to 5

times the sample concentrations (U.S. EPA, 1987e).

For ongoing assessment of method precision, it is recommended that one

laboratory duplicate and one matrix spike duplicate be analyzed with every 20

samples or with each sample batch, whichever is more frequent. In addition, it is

recommended that a laboratory control sample be analyzed at the above

frequency to allow an ongoing assessment of method performance, including an

estimate of method precision over time. Specific procedures for estimating

method precision by laboratory and/or matrix spike duplicates and laboratory

control samples are given in ASTM (1983). This reference also includes

procedures for estimating method precision from spike recoveries and for testing

for significant change in method precision over time.

Precision estimates obtained from the analysis of laboratory duplicates, matrix

spike duplicates, and repeated laboratory control sample analyses must fall within
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-38

specified control limits (see Table 8-6). If these values fall outside the control

limits, the analyses should be discontinued, appropriate corrective action taken,

and, if possible, the samples associated with the duplicates reanalyzed. If

reanalysis is not possible, all suspect data should be clearly identified.

Unacceptable precision estimates derived from the analysis of duplicate or

replicate samples may be caused by inadequate mixing of the sample before

aliquotting; inconsistent contamination; inconsistent digestion, extraction, or

cleanup procedures; or instrumentation problems (U.S. EPA, 1987e).

8.3.3.5.3 Routine assessment of analytical precision

The analysis of replicate aliquots of final sample extracts or digestates (analytical

replicates) provides an estimate of analytical precision only; it does not provide

an estimate of total method precision. For organic target analytes, analytical

replicates may be included at the discretion of the program manager or laboratory

supervisor. For the analysis of target metal analytes by graphite furnace atomic

absorption spectrophotometry (GFAA) and cold vapor atomic absorption

spectrophotometry (CVAA), it is recommended that duplicate injections of each

sample be analyzed and the mean concentration be reported. The RPD should

be within control limits established by the program manager or laboratory

supervisor, or the sample should be reanalyzed (U.S. EPA, 1987e).

8.3.3.5.4 Assessment of overall variability

Estimates of the overall variability of target analyte concentrations in a sample fish

or shellfish population and of the sampling and analysis procedures can be

obtained by collecting and analyzing field replicates. Replicate field samples are

optional in screening studies; however, if resources permit, it is recommended

that duplicate samples be collected at 10 percent of the screening sites as a

minimal QC check. Analysis of replicate field samples provides some degree of

variability in that the samples themselves are typically collected and exposed to

the same environmental conditions and contaminants. There are many points of

potential dissimilarity between samples of the type described here; however, this

variability is reduced when well-homogenized composite samples are analyzed.

In intensive studies, replicate samples should be collected at each sampling site

(see Section 6.1.2.7). Although the primary purpose of replicate field samples in

intensive studies is to allow more reliable estimates of the magnitude of

contamination, extreme variability in the results of these samples may also

indicate that sampling and/or analysis procedures are not adequately controlled.

8.3.3.6 Routine Monitoring of Interferences and Contamination—

Because contamination can be a limiting factor in the reliable quantitation of target

contaminants in tissue samples, the recommendations for proper materials and

handling and cleaning procedures given in Sections 6.2.2 and 7.2 should be

followed carefully to avoid contamination of samples in the field and laboratory.
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-39

Many metal contamination problems are due to airborne dust. High zinc blanks

may result from airborne dust or galvanized iron, and high chromium and nickel

blanks often indicate contamination from stainless steel. Mercury thermometers

should not be used in the field because broken thermometers can be a source of

significant mercury contamination. In the laboratory, samples to be analyzed for

mercury should be isolated from materials and equipment (e.g., polarographs)

that are potential sources of mercury contamination. Cigarette smoke is a source

of cadmium. Consequently, care should be taken to avoid the presence of

cigarette smoke during the collection, handling, processing, and analysis of

samples for cadmium. In organic analyses, phthalates, methylene chloride, and

toluene are common laboratory contaminants that are often detected in blanks at

concentrations above the MDL (U.S. EPA, 1987e).

Cross-contamination between samples should be avoided during all steps of

analysis of organic contaminants by GC-based methods. Injection micro-syringes

must be cleaned thoroughly between uses. If separate syringes are used for the

injection of solutions, possible differences in syringe volumes should be assessed

and, if present, corrected for. Particular care should be taken to avoid carryover

when high- and low-level samples are analyzed sequentially. Analysis of an

appropriate method blank may be required following the analysis of a high-level

sample to assess carryover (U.S. EPA, 1987e).

To monitor for interferences and contamination, the following blank samples

should be analyzed prior to beginning sample collection and analyses and on a

routine basis throughout each study (U.S. EPA, 1987e):

• Field blanks are rinsates of empty field sample containers (i.e., aluminum foil

packets and plastic bags) that are prepared, shipped, and stored as actual

field samples. Field blanks should be analyzed to evaluate field sample

packaging materials as sources of contamination. Each rinsate should be

collected and the volume recorded. The rinsate should be analyzed for target

analytes of interest and the total amount of target analyte in the rinsate

recorded. It is recommended that one field blank be analyzed with every 20

samples or with each batch of samples, whichever is more frequent.

• Processing blanks are rinsates of utensils and equipment used for dissecting

and homogenizing fish and shellfish. Processing blanks should be analyzed,

using the procedure described above for field blanks, to evaluate the efficacy

of the cleaning procedures used between samples. It is recommended that

processing blanks be analyzed at least once at the beginning of a study and

preferably once with each batch of 20 or fewer samples.

• Bottle blanks are rinsates of empty bottles used to store and ship sample

homogenates. Bottle blanks should be collected after the bottles are cleaned

prior to use for storage or shipment of homogenates. They should be

analyzed, using the procedure described above for field blanks, to evaluate

their potential as sources of contamination. It is recommended that one bottle
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-40

blank be analyzed for each lot of bottles or with each batch of 20 or fewer

samples, whichever is more frequent.

• Method blanks are samples of extraction or digestion solvents that are

carried through the complete analytical procedure, including extraction or

digestion; they are also referred to as procedural blanks. Method blanks

should be analyzed to evaluate contaminants resulting from the total analytical

method (e.g., contaminated glassware, reagents, solvents, column packing

materials, processing equipment). It is recommended that one method blank

be analyzed with every 20 samples or with each batch of samples, whichever

is more frequent.

• Reagent blanks are samples of reagents used in the analytical procedure.

It is recommended that each lot of analytical reagents be analyzed for target

analytes of interest prior to use to prevent a potentially serious source of

contamination. For organic analyses, each lot of alumina, silica gel, sodium

sulfate, or Florasil used in extract drying and cleanup should also be analyzed

for target analyte contamination and cleaned as necessary. Surrogate

mixtures used in the analysis of organic target analytes have also been found

to contain contaminants and the absence of interfering impurities should be

verified prior to use (U.S. EPA, 1987e).

Because the contamination in a blank sample may not always translate into

contamination of the tissue samples, analysts and program managers must use

their best professional judgment when interpreting blank analysis data. Ideally,

there should be no detectable concentration of any target analyte in any blank

sample (i.e., the concentration of target analytes in all blanks should be less than

the MDL). However, program managers may set higher control limits (e.g.,

MQL) depending on overall data quality requirements of the monitoring program.

If the concentration of a target analyte in any blank is greater than the established

control limit, all steps in the relevant sample handling, processing, and analysis

procedures should be reviewed to identify the source of contamination and

appropriate corrective action should be taken. If there is sufficient sample

material, all samples associated with the unacceptable blank should be

reanalyzed. If reanalysis is not possible, all suspect data should be identified

clearly.

Note: Analytical data should not be corrected for blank contamination by the

reporting laboratory; however, blank concentrations should always be reported

with each associated sample value.
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-41

8.3.3.7 Special QA and QC Procedures for the Analysis of Organic Target Analytes—

8.3.3.7.1 Routine monitoring of method performance

To account for losses during sample preparation (i.e., extraction, cleanup) and to

monitor overall method performance, a standard compound that has chemical and

physical properties as similar as possible to those of the target analyte of interest

should be added to each sample prior to extraction and to each calibration

standard. Such compounds may be termed surrogate recovery standards. A

stable, isotopically labeled analog of the target analyte is an ideal surrogate

recovery standard for GC/MS analysis.

If resources permit, an isotope dilution GC/MS technique such as EPA Method

1625 (40 CFR 136, Appendix A) is recommended for the analysis of organic

target analytes for which isotopically labeled analogs are available. In this

technique, RRFs used for quantitation may be calculated from measured isotope

ratios in calibration standards and not from instrument internal standards.

However, an instrument internal standard still must be added to the final sample

extract prior to analysis to determine the percent recoveries of isotopically labeled

recovery standards added prior to extraction. Thus, in isotope dilution methods,

instrument internal standards may be used only for QC purposes (i.e., to assess

the quality of data) and not to quantify analytes. Control limits for the percent

recovery of each isotopically labeled recovery standard should be established by

the program manager, consistent with program data quality requirements. Control

limits for percent recovery and recommended corrective actions given in EPA

Method 1625 (40 CFR 136, Appendix A) should be used as guidance.

If isotopically labeled analogs of target analytes are not available or if the isotope

dilution technique cannot be used (e.g., for chlorinated pesticides and PCBs

analyzed by GC/ECD), other surrogate compounds should be added as recovery

standards to each sample prior to extraction and to each calibration standard.

These surrogate recovery standards should have chemical and physical

properties similar to the target analytes of interest and should not be expected to

be present in the original samples. Recommended surrogate recovery standards

are included in the methods referenced in Table 8-2 and in EMMI (U.S. EPA,

1991f). 

Samples to which surrogate recovery standards have been added are termed

surrogate spikes. The percent recovery of each surrogate spike (% Rs) should

be determined for all samples as follows:

% Rs = 100 (Cm/Ca) (8-10)

where

% Rs = Surrogate spike percent recovery

Cm = Measured concentration of surrogate recovery standard
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-42

Ca = Actual concentration of surrogate recovery standard added to the

sample.

Control limits for the percent recovery of each surrogate spike should be

established by the program manager consistent with program data quality

requirements. The control limits in the most recent EPA CLP methods (U.S.

EPA, 1991c) are recommended for evaluating surrogate recoveries.

Note: Reported data should not be corrected for percent recoveries of surrogate

recovery standards. Recovery data should be reported for each sample to

facilitate proper evaluation and use of the analytical results.

8.3.3.7.2 Other performance evaluation procedures

The following additional procedures are required to evaluate the performance of

GC-based analytical systems prior to the routine analysis of field samples (U.S.

EPA, 1989c; U.S. EPA, 1991c). It is the responsibility of each program manager

to determine specific evaluation procedures and control limits appropriate for their

data quality requirements.

Evaluation of the GC system

GC system performance should be evaluated by determining the number of

theoretical plates of resolution and the relative retention times of the internal

standards.

Column Resolution: The number of theoretical plates of resolution, N, should

be determined at the time the calibration curve is generated (using

chrysene-d10) and monitored with each sample set. The value of N should not

decrease by more than 20 percent during an analysis session. The equation

for N is given as follows:

N = 16 (RT/W)2 (8-11)

where

RT = Retention time of chrysene-d10 (s)

W = Peak width of chrysene-d10 (s).

Relative Retention Time: Relative retention times of the internal standards

should not deviate by more than ±3 percent from the values calculated at the

time the calibration curve was generated.

If the column resolution or relative retention times are not within the specified

control limits, appropriate corrective action (e.g., adjust GC parameters, flush GC

column, replace GC column) should be taken.
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-43

Evaluation of the MS system

The performance of the mass spectrometer should be evaluated for sensitivity

and spectral quality.

Sensitivity: The signal-to-noise value should be at least 3.0 or greater for m/z

198 from an injection of 10 ng decafluorotriphenylphosphine (DFTPP).

Spectral Quality: The intensity of ions in the spectrum of a 50-ng injection of

DFTPP should meet the following criteria (U.S. EPA, 1991c):

m/z Criteria

51 30-80% mass 198

68 <2% mass 69

69 present

70 <2% mass 69

127 25-75% mass 198

197 <1% mass 198

198 base peak, 100% relative abundance

199 5-9% mass 198

275 10-30% mass 198

365 >0.75% mass 198

441 present and <mass 443

442 40-110% mass 198

443 15-24% mass 442

If the control limits for sensitivity or spectral quality are not met, appropriate

corrective action (e.g., clean MS, retune MS) should be taken.

Evaluation of cleanup columns

Because the fatty content of many tissue samples may overload the cleanup

columns, these columns should be calibrated and monitored regularly to ensure

that target analytes are consistently collected in the proper fraction. Gel

permeation columns should be monitored by visual inspection (for column

discoloration, leaks, cracks, etc.) and by measurement of flow rate, column

resolution, collection cycle, and method blanks (see Section 8.3.3.6). Silica gel

columns should be evaluated by their ability to resolve cholesterol from a selected

target analyte.

8.3.3.8 External QA Assessment of Analytical Performance—

Participation in an external QA program by all analytical laboratories in state fish

and shellfish consumption advisory programs is strongly recommended for

several reasons:
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-44

• To demonstrate laboratory capability prior to conducting routine analyses of

field samples

• To provide an independent ongoing assessment of each laboratory's

capability to perform the required analyses

• To enhance the comparability of data between states and Regions.

Two types of external QA programs are recommended: round-robin interlaboratory comparisons (often referred to as interlaboratory calibration programs)

and split-sample interlaboratory comparisons.

8.3.3.8.1 Round-robin analysis interlaboratory comparison program

At present, the only external round-robin QA program available for analytical

laboratories conducting fish and shellfish tissue analyses for environmental

pollutants is administered by NOAA in conjunction with its National Status and

Trends (NS&T) Program (Cantillo, 1991). This QA program has been designed

to ensure proper documentation of sampling and analysis procedures and to

evaluate both the individual and collective performance of participating

laboratories. Recently, NOAA and EPA have agreed to conduct the NS&T

Program and the EMAP-NC Program as a coordinated effort. As a result, EMAPNC now cosponsors and cooperatively funds the NS&T QA Program, and the

interlaboratory comparison exercises include all EMAP-NC laboratories (U.S.

EPA, 1991e).

Note: Participation in the NS&T QA program by all laboratories performing

chemical analyses for state fish and shellfish contaminant monitoring programs

is recommended to enhance the credibility and comparability of analytical data

among the various laboratories and programs.

Each laboratory participating in the NS&T QA program is required to demonstrate

its analytic capability prior to the analysis of field samples by the blind analysis of

a fish and shellfish tissue sample that is uncompromised, homogeneous, and

contains the target analytes of interest at concentrations of interest. A

laboratory's performance generally will be considered acceptable if its reported

results are within ±30 percent (for organics) and ±15 percent (for metals) of the

actual or certified concentration of each target analyte in the sample (U.S. EPA,

1991e). If any of the results exceed these control limits, the laboratory will be

required to repeat the analysis until all reported results are within the control

limits. Routine analysis of field samples will not be allowed until initial

demonstration of laboratory capability is acceptable.

Following the initial demonstration of laboratory capability, each participating

laboratory is required to participate in one intercomparison exercise per year as

a continuing check on performance. This intercomparison exercise includes both

organic and inorganic (i.e., trace metals) environmental and standard reference
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-45

samples. The organic analytical intercomparison program is coordinated by NIST,

and the inorganic analytical intercomparison program is coordinated by the

NRCC. Sample types and matrices vary yearly. Performance evaluation samples

used in the past have included accuracy-based solutions, sample extracts, and

representative matrices (e.g., tissue or sediment samples). Laboratories are

required to analyze the performance evaluation samples blind and to submit their

results to NIST or NRCC, as instructed. Individual laboratory performance is

evaluated against the consensus values (i.e., grand means) of the results

reported by all participating laboratories. Laboratories that fail to achieve

acceptable performance must take appropriate corrective action. NIST and

NRCC will provide technical assistance to participating laboratories that have

problems with the intercomparison analyses. At the end of each calendar year,

the results of the intercomparison exercises are reviewed at a workshop

sponsored by NIST and NRCC. Representatives from each laboratory are

encouraged to participate in these workshops, which provide an opportunity for

discussion of analytical problems encountered in the intercomparison exercises.

Note: Nonprofit laboratories (e.g., EPA and other federal laboratories, state,

municipal, and nonprofit university laboratories) may participate in the NS&T QA

program at no cost on a space-available basis. The cost of participation in the

NIST Intercomparison Exercise Program for Organic Contaminants in the Marine

Environment is $2,500 for private laboratories within and outside the United

States. This cost covers samples for one exercise per year. Samples may be

obtained directly from NIST by contacting Ms. Michele Shantz, NIST, 100 Bureau

Drive, Stop 8392, Gaithersburg, MD 20899-8392; Tel: 301-975-3106, FAX:

301-997-0685. Trace inorganic samples are available directly from NRCC by

contacting Mr. Scott Willis, NRCC, Ottawa, Ontario, Canada K1A029, e-mail:

scott.willie@NRC.CA, Tel: 613-993-4969.

To obtain additional information about participation in the NS&T QA program,

contact Dr. Adriana Cantillo, QA Manager, NOAA/National Status and Trends

Program, NYSCI1, 1305 East West Highway, Silver Spring, MD 20910;

Tel: 301-713-3028, ext. 147, FAX: 301-713-4388.

8.3.3.8.2 Split sample analysis interlaboratory comparison programs

Another useful external QA procedure for assessing interlaboratory comparability

of analytical data is a split-sample analysis program in which a percentage

(usually 5 to 10 percent) of all samples analyzed by each state or Region are

divided and distributed for analyses among laboratories from other states or

Regions. Because actual samples are used in a split-sample analysis program,

the results of the split-sample analyses provide a more direct assessment of the

comparability of the reported results from different states or Regions.

The NS&T QA program does not include an interlaboratory split-sample analysis

program. However, it is recommended that split-sample analysis programs be

established by states and/or Regions that routinely share results. 
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-46

8.4 Documentation and Reporting of Data

The results of all chemical analyses must be documented adequately and

reported properly to ensure the correct evaluation and interpretation of the data.

8.4.1 Analytical Data Reports

The documentation of analytical data for each sample should include, at a

minimum, the following information:

• Study identification (e.g., project number, title, phase)

• Description of the procedure used, including documentation and justification

of any deviations from the standard procedure

• Method detection and quantitation limits for each target analyte

• Method accuracy and precision for each target analyte

• Discussion of any analytical problems and corrective action taken

• Sample identification number

• Sample weight (wet weight)

• Final dilution volume/extract volume

• Date(s) of analysis

• Identification of analyst

• Identification of instrument used (manufacturer, model number, serial number,

location)

• Summary calibration data, including identification of calibration materials,

dates of calibration and calibration checks, and calibration range(s); for

GC/MS analyses, include DFTPP spectra and quantitation report

• Reconstructed ion chromatograms for each sample analyzed by GC/MS

• Mass spectra of detected target compounds for each sample analyzed by

GC/MS

• Chromatograms for each sample analyzed by GC/ECD and/or GC/FID

• Raw data quantitation reports for each sample
8. LABORATORY PROCEDURES II — SAMPLE ANALYSES

8-47

• Description of all QC samples associated with each sample (e.g., reference

materials, field blanks, rinsate blanks, method blanks, duplicate or replicate

samples, spiked samples, laboratory control samples) and results of all QC

analyses. QC reports should include quantitation of all target analytes in each

blank, recovery assessments for all spiked samples, and replicate sample

summaries. Laboratories should report all surrogate and matrix spike recovery

data for each sample; the range of recoveries should be included in any

reports using these data.

• Analyte concentrations with reporting units identified (as ppm or ppb wet

weight, to two significant figures unless otherwise justified). Note: Reported

data should not be recovery- or blank-corrected.

• Lipid content (as percent wet weight)

• Specification of all tentatively identified compounds (if requested) and any

quantitation data.

• Data qualifications (including qualification codes and their definitions, if

applicable, and a summary of data limitations).

To ensure completeness and consistency of reported data, standard forms should

be developed and used by each laboratory for recording and reporting data from

each analytical method. Standard data forms used in the EPA Contract

Laboratory Program (U.S. EPA, 1991b, 1991c) may serve as useful examples for

analytical laboratories.

All analytical data should be reviewed thoroughly by the analytical laboratory

supervisor and, ideally, by a qualified chemist who is independent of the

laboratory. In some cases, the analytical laboratory supervisor may conduct the

full data review, with a more limited QA review provided by an independent

chemist. The purpose of the data review is to evaluate the data relative to data

quality specifications (e.g., detection and quantitation limits, precision, accuracy)

and other performance criteria established in the Work/QA Project Plan. In many

instances, it may be necessary to qualify reported data values; qualifiers should

always be defined clearly in the data report. Recent guidance on the

documentation and evaluation of trace metals data collected for Clean Water Act

compliance monitoring (U.S. EPA, 1995h) provides additional useful information

on data review procedures.

8.4.2 Summary Reports

Summaries of study data should be prepared for each target species at each

sampling site. Specific recommendations for reporting data for screening and

intensive studies are given in Section 9.2.
9. DATA ANALYSIS AND REPORTING

9-1

SECTION 9

DATA ANALYSIS AND REPORTING

This section provides guidance on (1) analysis of laboratory data for both

screening and intensive studies that should be included in state data reports, (2)

data reporting requirements for both state-conducted screening and intensive

studies, and (3) data reporting requirements for a national data repository for

state-collected fish tissue data housed within the National Listing of Fish and

Wildlife Advisories (NLFWA) database. 

All data analysis and reporting procedures should be documented fully as part of

the Work/QA Project Plan for each study, prior to initiating the study (see

Appendix I). All routine data analysis and reporting procedures should be

described in standard operating procedures. In particular, the procedures to be

used to determine if the concentration of a target analyte in fish or shellfish tissue

differs significantly from the selected screening value must be clearly

documented.

9.1 DATA ANALYSIS

9.1.1 Screening Studies

The primary objective of Tier 1 screening studies is to assist states in identifying

potentially contaminated harvest areas where further investigation of fish and

shellfish contamination may be warranted. The criteria used to determine whether

the measured target analyte concentration in a fish or shellfish tissue composite

sample is different from the SV (greater than or less than) should be clearly

documented. If a reported target analyte concentration exceeds the SV in the

screening study, a state should initiate a Tier 2, Phase I, intensive study (see

Section 6.1.2.1) to verify the level of contamination in the target species. Because

of resource limitations, some states may choose to conduct a risk assessment

using screening study data; however, this approach is not recommended because

a valid statistical analysis cannot be performed on a single composite sample. If

a reported analyte concentration is close to the SV but does not exceed the SV,

the state should reexamine historic data on water, sediment, and fish tissue

contamination at the site and evaluate data on laboratory performance. If these

data indicate that further examination of the site is warranted, the state should

initiate a Tier 2, Phase I, intensive study to verify the magnitude of the

contamination.
9. DATA ANALYSIS AND REPORTING

9-2

Because replicate composite samples are not required as part of a screening

study, estimating the variability of the composite target analyte concentration at

any site is precluded. The following procedure is recommended for use by states

for analysis of the individual target analyte concentration for each composite

sample from reported laboratory data (see Section 8.3.3.3)

• A datum reported below the method detection limit, including a datum reported

as not detected (i.e., ND, no observed response) should be assigned a value

of one-half the MDL or zero.

• A datum reported between the MDL and the method quantitation limit should

be assigned a value of the MDL plus one-half the difference between the MQL

and the MDL.

• A datum reported at or above the MQL should be used as reported.

This approach is similar to that published in 40 CFR Parts 122, 123, 131, and

132—Proposed Water Quality Guidance for the Great Lakes System.

If resources permit and replicate composite samples are collected at a suspected

site of contamination, then a state may conduct a statistical analysis of differences

between the mean target analyte concentration and the SV, as described in

Section 9.1.2.

9.1.2 Intensive Studies

The primary objectives of Tier 2 intensive studies are to confirm the findings of the

screening study by assessing the magnitude and geographic extent of the

contamination in various size classes of selected target species. The EPA Office

of Water recommends that states collect replicate composite samples of three

size classes of each target species in the study area to verify whether the mean

target analyte concentration of replicate composite samples for any size class

exceeds the SV for any target analyte identified in the screening study. The

statistical approach for this comparison is described in Section 6.1.2.7.

The following procedure is recommended for use by states in calculating the

mean arithmetic target analyte concentration from reported laboratory data (see

Section 8.3.3.3.3).

• Data reported below the MDL, including data reported as not detected (i.e.,

ND, no observed response) should be assigned a value of one-half the MDL.

• Data reported between the MDL and the MQL should be assigned a value of

the MDL plus one-half the difference between the MQL and the MDL.

• Data reported at or above the MQL should be used as reported.

This approach is similar to that published in 40 CFR Parts 122, 123, 131, and

132—Proposed Water Quality Guidance for the Great Lakes System.

Secondary objectives that may be assessed as part of Tier 2 intensive studies
9. DATA ANALYSIS AND REPORTING

9-3

can include defining the geographical region where fish contaminant concentrations exceed screening values; identifying geographical distribution of contaminant

concentrations; and, in conjunction with historical data or future data collection,

assessing changes in fish contaminant concentrations over time. The statistical

considerations involved in comparing fish contaminant levels measured at

different locations or times are discussed in Appendix N.

State staff should consult a statistician in interpreting intensive study tissue

residue results to determine the need for additional monitoring, risk assessment,

and issuance of a fish or shellfish consumption advisory. Additional information

on risk assessment, risk management, and risk communication procedures will

be provided in later volumes in this guidance series (see Section 1.4).

9.2 DATA REPORTING

9.2.1 State Data Reports

State data reports should be prepared by the fish contaminant monitoring program

manager responsible for designing the screening and intensive studies.

Summaries of Tier 1 screening study data should be prepared for each target

species sampled at each screening site. For Tier 2 intensive studies (Phase I

and Phase II), data reports should be prepared for each target species (by size

class, as appropriate) at each sampling site within the waterbody under

investigation (see Section 6.1.2). Screening and intensive study data reports

should include, at a minimum, the information shown in Figure 9-1.

9.2.2 Reports to the National Fish Tissue Residue Data Repository (NFTRDR)

The EPA Office of Science and Technology within the Office of Water has established the NFTRDR, which is housed within the NLFWA database. This repository

is a collection of fish and shellfish contaminant monitoring data gathered by

various state, federal, and local agencies for advisory purposes. The objectives

of the repository are to:

• Facilitate the exchange of fish and shellfish contaminant monitoring data

nationally by improving the comparability and integrity of state data

• Encourage greater cooperation among regional and state fish advisory

programs

• Assist states in their fish tissue data collection efforts by providing ongoing

technical assistance.

The NLFWA database now contains a facility for storing fish tissue residue data

as well as for documenting and mapping active and rescinded fish consumption

advisories. Since 1996, a stand-alone version of the NFLWA database has been

available for Internet downloads. Internet WEB-based tools have recently been

developed to support queries and interactive mapping of both the general advisory

information as well as fish tissue residue data. Internet-based tools are also being
9. DATA ANALYSIS AND REPORTING

9-4

• Study identification (e.g., project number, title, and study type)• Program manager• Sampling site name• Latitude (decimal degrees preferred)• Longitude (decimal degrees preferred)• Type of waterbody (lake, river, estuary, etc.)• Name of waterbody• Sampling date (e.g., YYYYMMDD, Year 2000 compliant format)• Sampling time (e.g., HH, MM in a 24-h format)• Sampling gear type used (e.g., dredge, seine, trawl, gill net)• Sampling depth (feet or meters)• Standard common name of target species (preferably name

sanctional by American Fisheries Society for inland waters or byNOAA for coastal waters)

• Composite sample numbers• Number of individuals in each composite sample• Number of replicate composite samples• Predominant characteristics of specimens used in each composite

sample- Predominant life stage of individuals in composite- Predominant sex of individuals in composite (if applicable)- Average age of individuals in composite (if applicable)- Average body length (cm)- Description of edible portion (tissue type)

• Analytical methods used (including method for lipid analysis)• Method detection and quantitation limits for each target analyte

analyzed

• Sample cleanup procedures (e.g., additional steps taken to further

purify the sample extracts or digestates)

• Data qualifiers (e.g., additional qualifying information about the

measurement) 

• Percent lipid (wet weight basis) in each composite sample• For each target analyte in each composite sample:

- Total wet weight of composite sample (g) used in analysis- Measured concentration (wet weight basis) as reported by the

laboratory (see Section 8.3.3.3.3)

- Units of measurement for target analyte concentration (e.g., ppm)- Evaluation of laboratory performance (i.e., description of all QA

and QC samples associated with the sample(s) and results of allQA and QC analyses)

• In screening studies with only one composite sample for each target

species, the state should provide for each target analyte acomparison of reported concentration with selected SV (seeSection 4 tables) and indication of whether SV was exceeded forrecreational or subsistence fishers (see Section 9.1.1).

• In intensive studies, for each target analyte in each set of replicate

composite samples, the state should provide- Range of target analyte concentrations for each set of replicate

composite samples

- Mean (arithmetic) target analyte concentration for each set of

replicate composite samples (see Section 9.1.2)

- Standard deviation of mean target analyte concentration- Comparison of target analyte arithmetic mean concentration with

selected SV (see Section 5) and indication of whether SV wasexceeded for recreational or subsistence fishers

Figure 9-1. Recommended data reporting requirements for screening and intensive studies.
9. DATA ANALYSIS AND REPORTING

9-5

developed as a way for state agencies to add fish advisory and contaminant

monitoring data to the NLFWA database and may be developed to perform some

types of standard data analysis on the fish tissue residue data. 

EPA has recently developed an Internet-based data entry facility for the NLFWA

using some of the data elements included in Figure 9-1. This Internet-based data

entry facility is housed within the EPA’s NLFWA database and allows states to

archive fish advisory information as well as fish tissue residue data generated

through their fish contaminant monitoring programs. States may prepare their

own data tables and arrange to transfer these to EPA to be formatted and

reviewed before entry into the repository. The information in the NFTRDR can be

organized into three different tables (STATIONS, SAMPLES, and RESULTS

tables) using such readily available PC relational database packages as

ACCESS (Figure 9-2). If states submit their monitoring data in other file formats

(e.g., spreadsheet files or ASCII files exported from other in-house database

systems), a short data dictionary (metadata) file should be included (ASCII,

Wordperfect, or WORD format) clearly documenting the meaning of all data fields

and any codes, abbreviations, or measurement units used in the files.

State, regional, and local agency staff may obtain further information on the new

Internet WEB-based database EPA now has available by contacting:

U.S. Environmental Protection Agency

Office of Science and Technology

National Fish and Wildlife Contamination Program-4305

1200 Pennsylvania Avenue, NW

Washington, DC 20460

PHONE: 202-260-7301

FAX: 202-260-9830

Jeffrey D. Bigler

U.S. Environmental Protection Agency-4305

1200 Pennsylvania Avenue, NW

Washington, DC 20460

PHONE: 202-260-1305

E-MAIL: bigler.jeff@epa.gov
9. DATA ANALYSIS AND REPORTING

9-6

Fish Tissue Chemical Residue Data Tables: STATIONS, SAMPLES and RESULTS

The STATIONS table includes basic locational data.

Field name Field description

STATION_ID Waterbody, Station or Monitoring Site Identifier. This field becomes a database key

field. Each record must have a unique STATION_ID.

STATE State 2-character postal code abbreviation.

WATERBODY (or SITENAME) A short caption to identify the waterbody or sampling station.

LOCATION Additional descriptive information on the waterbody or station location.

ADVNUM If the waterbody or site is associated with an advisory (active or rescinded), include

the number assigned to this advisory in the current National Listing of Fish and

Wildlife Advisories (NLFWA) database.

COUNTY County name.

LAT Station latitude. A format in decimal degrees is preferred.

LNG Station longitude. A format in decimal degrees is preferred.

The SAMPLES table includes data on the type of tissue sample collected.

Field name Field description

SAMPLE_ID An identifier to each specific fish tissue sample from a waterbody or station. This is

used as a database key, so each record must have a unique SAMPLE_ID

STATION_ID Waterbody, Station or Monitoring Site Identifier as defined in the STATIONS table.

SAMPLE_DATE The date the sample was collected in the field. Give date in a Year 2000 compliant

format (YYYYMMDD).

FISH_SPECIES Fish species names. Standard English common names as established by the

American Fisheries Society for inland waters or NOAA for coastal water are

preferred.

SAMPLE_TYPE How the sample was prepared (e.g., fillet with skin-on or skin-off, whole fish). In the

NUMBER_OF_FISH field below, multiple fish in a sample indicate a composite

sample.

LENGTH The length of the sample fish. For composites, an average length should be given.

LENGTH_UNIT Length units of fish (cm or inches)

WEIGHT Specimen or composite weight used for residue analysis. 

WEIGHT_UNIT Weight units (usually in grams).

LIPID Percent extractable lipids.

NUMBER_OF_FISH Number of fish (specimens) in sample. Number greater than a value of 1 indicates

a composite sample.

The RESULTS table includes chemical-specific tissue sample concentrations.

Field name Field description

SAMPLE_ID An identifier to each specific fish tissue sample from a waterbody or station. This

is used as a database key, so each record must have a unique SAMPLE_ID

PARAMETER Chemical name. File should specify all acronyms or abbreviations used.

DETECTION_INFO A caption to document detection limit information (e.g., “less than detection limit”).

RESULT A number representing the concentration of a chemical (or the detection limit).

RESULT_UNIT Units associated with concentration (e.g., “ppm”).

Figure 9-2. Key information fields for the National Fish Tissue Residue Data Repository.
10. LITERATURE CITED

10-1

SECTION 10

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10. LITERATURE CITED

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APPENDIX A

1993 FISH CONTAMINANT WORKGROUP
APPENDIX A

A-3

A. 1993 FISH CONTAMINANT WORKGROUP

These individuals representing EPA Headquarters, EPA Regions, State and

Federal agencies, Native American groups and others provided technical

information, reviews, and recommendations throughout the preparation of the first

edition of this document. Participation in the review process does not imply

concurrence by these individuals with all concepts and methods described in this

document.

A.1 EPA Headquarters Staff

Charles Abernathy EPA/Office of Water

Thomas Armitage EPA/Office of Water

Jeffrey Bigler EPA/Office of Water (Workgroup Chairman)

Carin Bisland EPA/Office of Water

Dennis Borum EPA/Office of Water

Robert Cantilli EPA/Office of Water

Julie Du EPA/Office of Water

Richard Hoffman EPA/Office of Water

Clyde Houseknecht EPA/Office of Water

Henry Kahn EPA/Office of Water

Amal Mahfouz EPA/Office of Water

Michael Kravitz EPA/Office of Water

Elizabeth Southerland EPA/Office of Water

Margaret Stasikowski EPA/Office of Water

Irene Suzukida-Horner EPA/Office of Water

Elizabeth Tam EPA/Office of Water

William Telliard EPA/Office of Water

Charles White EPA/Office of Water

Jennifer Orme Zavala EPA/Office of Water

Tina Levine EPA/Office of Pesticide Programs

Michael Metzger EPA/Office of Pesticide Programs

Richard Whiting EPA/Office of Pesticide Programs

Jacqueline Moya EPA/Office of Health and Environmental

Assessment

A.2 Other EPA Office Staff

David DeVault EPA/Great Lakes National Program Office

Brian Melzian EPA/Office of Reserach and DevelopmentNarragansett, RI

John Paul EPA/Office of Research and DevelopmentNarragansett, RI

Dennis McMullen EPA/Environmental Monitoring and

Systems Laboratory-Cincinnati, OH

Laurence Burkhard EPA/Office of Research and DevelopmentDuluth, MN
APPENDIX A

A-4

Michael Dourson EPA/Office of Health and Environmental 

Assessment-Cincinnati, OH

Donald Klemm EPA/Office of Health and Environmental

Assessment-Cincinnati, OH

A.3 EPA Regional Staff

Charles Kanetsky Region 3

Jerry Stober Region 4

Peter Redmon Region 5

Diane Evans Region 6

Philip Crocker Region 7

Bruce Herbold Region 9

A.4 Other Federal Agency Staff

Michael Bolger FDA

Leon Sawyer FDA

Lee Barclay FWS

Frank De Luise FWS

Donald Steffeck FWS

Jerry Schulte ORSANCO

Adriana Cantillo NOAA

Maxwell Eldridge NOAA

Betty Hackley NOAA

Alicia Jarboe NOAA

Bruce Morehead NOAA

Don Dycus TVA

J. Kent Crawford USGS

A.5 State Agency Staff

Robert Cooner Alabama

Brian Hughes Alabama

William Keith Arkansas

Thomas McChesney Arkansas

Randall Mathis Arkansas

Gerald Pollock California

Robert McConnell Colorado

Richard Greene Delaware

Eldert Hartwig Florida

Randall Manning Georgia

Robert Flentge Illinois

C. Lee Bridges Indiana

Emelise Cormier Louisiana

Albert Hindrichs Louisiana

Elaine Sorbet Louisiana
APPENDIX A

A-5

Deirdre Murphy Maryland

Jack Schwartz Massachusetts

John Hesse Michigan

Richard Powers Michigan

Lisa Williams Michigan

Pamela Shubat Minnesota

Alan Buchanan Missouri

David Tunink Nebraska

Donald Normandeau New Hampshire

Paul Hauge New Jersey

Lawrence Skinner New York

Ken Eagleson North Carolina

Jay Sauber North Carolina

Luanne Williams North Carolina

Michael Ell North Dakota

Martin Schock North Dakota

Abul Anisuzzaman Ohio

Gene Foster Oregon

Barbara Britton Texas

Peter Sherertz Virginia

Ram Tripathi Virginia

Jim Amrhein Wisconsin

Bruce Baker Wisconsin

A.6 Other Organizations

James Wiener American Fisheries Society

Deborah Schwackhamer University of Minnesota

Alvin Braswell North Carolina State Museum of Natural

Science

J. Whitfield Gibbons University of Georgia Savannah River

Ecology Laboratory
APPENDIX B

SCREENING VALUES FOR DEFINING GREEN AREAS
APPENDIX B

B-3

APPENDIX B

Recommendations for Designating Areas of Unrestricted Fish 

Consumption as Part of State Fish Advisory Programs 

Fish consumption advisories issued across the United States have increased over

the past 5 years from 1,266 advisories in 1993 to 2,506 advisories in 1998. Many

states are now advising their citizens either (1) not to consume any fish or any fish

of a specific species or specific size class from specified waterbodies, or (2) to

restrict their consumption of these fish to a specified number of meals per time

interval (such as one meal per week or two meals per month). This comes at a

time when the health benefits of consuming fish have also become widely

recognized (Burr et al., 1989; Dolecek and Granditis, 1991; Kimbrough, 1991;

Knapp and Fitzgerald, 1989; Kromhout, 1993; Kromhout et al., 1985; McVeigh,

1990; Norell et al.,1986; Shekelle et al., 1985; Simopoulous, 1991). In an attempt

to promote consumption of fish with relatively low body burdens of chemical

contaminants as part of a healthy diet, some states have defined certain

waterbodies as containing fish that are safe for “unrestricted consumption.”

These areas that are identified as safe for unrestricted fish consumption are often

referred to as “green” areas. The U.S. Environmental Protection Agency (EPA)

National Fish and Wildlife Contamination Program is recommending that states

develop an approach for designating and communicating the location of these

safe fishing areas to the fish-consuming public. This risk management tool

encourages both fishing as a recreational activity and the consumption of fish that

are low in chemical contaminant residues, high in protein, and low in fat content.

This green area concept, already in use in several U.S. states and Canadian

provinces, would enable states, territories, and tribes to define areas where fish

tissue monitoring data and appropriate risk assessments have determined that

fish may be safely consumed at unrestricted levels (as defined by the state) from

a particular waterbody or waterbodies in a particular watershed. The green areas

concept is in contrast to the more traditionally issued fish advisory that

discourages fish consumption from specified waterbodies altogether or advises

reduced consumption of fish. These green areas may comprise watersheds that

are relatively undeveloped from an industrial and agricultural perspective, such

as wilderness areas, or areas that border county, state, or national forests or

preserves. One cautionary note with regard to waterbodies in very remote areas

must be made, however. Several studies have monitored what were perceived as

pristine watersheds and unexpectedly found elevated chemical contamination in

fish tissues at levels of potential human health concern (Datta et al., 1999; Grieb

et al., 1990; Henry et al., 1998; Sorensen et al., 1990; Swackhamer and Hites,

1998). Although these waterbodies were removed from direct industrial point

source discharges and agricultural nonpoint source pollution, several chemical

contaminants such as mercury, toxaphene, and PCBs, can be transported in the

atmosphere from highly contaminated areas and be deposited relatively long

distances from the actual pollutant sources. This atmospheric transportation of
APPENDIX B

B-4

some chemical contaminants has resulted in the issuance of statewide freshwater

advisories for mercury in 10 states (U.S. EPA, 2000). 

Most states sample fish from a variety of waters during their annual fish

monitoring programs. Not all waterbodies sampled are found to be contaminated

to such a degree that issuance of advisories is necessary. It is those

waterbodies containing fish with lower chemical residues (below human health

screening values) that would potentially fall under the broad category of green

areas. Within the green areas, however, there need to be criteria for

distinguishing those waterbodies that are only slightly below the human health

levels of concern from those that are truly pristine with respect to chemical

contaminant levels in fish tissues. Once these green areas have been identified,

states can use appropriate information on fish-consuming populations to

establish appropriate consumption information.

To designate a waterbody as a green area where unrestricted fish consumption

(as defined by the state) is sanctioned, EPA recommends that a state

 Collect a variety of fish species in the waterbody under review for green area

status, but particularly target those species that are generally consumed by

the local recreational or subsistence fishers using the waterbody. 

 Assess levels of contamination for all of the 25 target analytes identified in

this guidance document in the sampled fish tissue that are likely to impact

that waterbody and compare residue levels to selected human health

screening values.

 Conduct a risk assessment of the resulting chemical analysis data to

determine whether the waterbody can be designated a green area and to

more clearly define “unrestricted consumption” for the fish-consuming

population given the specific levels of contamination for each of the target

analytes found

 Clearly define for and communicate to the fish-consuming public the

definition of “unrestricted consumption” based on the specific assumption

used in the risk assessment procedure for the green area waters so that all

segments of the fish-consuming public including sensitive populations (e.g.,

pregnant women/fetuses, nursing mothers, and children) understand the

limitations of this unrestricted consumption status.

EPA suggests that the states follow the guidance in this volume for designing a

monitoring program (Sections 2, 3, and 6), including the selection and sampling

of appropriate target species in adequate numbers and of appropriate size

classes. Two distinct screening values are available to the states based on

different consumption rates of two distinct fisher populations: recreational fishers

and subsistence fishers. State-collected information from creel surveys or

interviews with these two distinct populations is most desirable for use in deriving
APPENDIX B

B-5

screening values. (See U.S. EPA, 1998, Guidance for Conducting Fish and

Wildlife Consumption Surveys for further information.) If local information on

these fisher populations is not available, states may use the EPA default

consumption rate values of 17.5 g/d and 142.4 g/d for recreational and

subsistence populations, respectively, to calculate screening values. 

Table B-1 summarizes the screening values (SVs) that states may choose to use

to initially identify green areas. Screening values for the 25 target analytes are

provided for both recreational and subsistence fishers based on the EPA default

consumption rates (see Tables 5-3 and 5-4 in Section 5.2 for additional

information on calculating screening values.) These calculated SVs for each of

the target analytes should not be exceeded in fish tissues for the respective

target fish-consuming population. The SVs listed in the table for target analytes

such as inorganic arsenic, chlordane, DDT, dieldrin, heptachlor epoxide,

hexachlorobenzene, lindane, toxaphene, oxyfluorfen, and PCBs that have both

noncancer and cancer health endpoints are represented by the more

conservative or protective of the two calculated SVs.

One concern states must address relates to the detection limit of the analytical

method selected for chemical analysis of each target analyte in fish tissue

samples. Just because an analyte cannot be detected in fish tissue, does not

ensure that the area is safe for unrestricted consumption. For some of the target

analytes, especially those calculated using subsistence consumption rates, the

SVs are at or below the detection limit for even the most state-of-the-art residue

analysis methods (see Table B-1). Thus, the analytical result of a sample being

less than the mean detection limit for a particular analyte will not provide the

state with adequate information about the actual contaminant level to accurately

determine the meal size and meal frequency that can safely be consumed.

States in many cases have been forced by limited monitoring resources to target

the collection and analysis of fish tissues to those waterbodies deemed most

likely to be contaminated by chemical pollutants. Unlike sampling to determine

whether a fish consumption advisory should be issued for a chemical

contaminant, which requires only that one chemical be found in exceedance of

a human health SV, sampling and analysis to determine green area status must

confirm that there are no chemical contaminants in exceedance of the selected

human health SVs. It is also important that the state directly monitor the

contaminant tissue levels of the various chemical contaminants of concern in fish

tissue rather than rely on indirect methods such as measuring water or sediment

contaminant levels to estimate the level of fish tissue contamination in a

particular waterbody. 

EPA further recommends that states clearly define for the fish-consuming

population the meaning of “unrestricted consumption.” For example, a state may

choose a green area designation for their jurisdictional waters that are primarily

used by recreational fishers. These waters must then not exceed SVs for
APPENDIX B

B-6

Table B-1. Screening Values for Defining Green Areas Based on

Recreational or Subsistence Use of the Waterbody (ppm)

Target Analyte

SV Based on

Recreational

Fisher

Consumption

SV Based on

Subsistence

Fisher

Consumption

Analytical

Detection 

Limits

Arsenic (inorganic) 0.026 3.87 x 10-3 a 0.005

Cadmium 4 0.58 0.005

Mercury 0.4 5.8 x 10-2 0.001

Selenium 20 2.9 0.017

Tributyltin 1.2 0.17 0.002

Chlordane (Total) 0.114 0.016 0.001

DDT (Total) 0.117 0.017 1 x 10-4

Dicofol 1.6 0.23 0.001

Dieldrin 2.5 x 10-3 3.07 x 10-4 a 1 x 10-4

Endosulfan (I and II) 24 2.949 0.005

Endrin 1.2 0.147 1 x 10-4

Heptachlor epoxide 4.39 x 10-3 5.40 x 10-4 a 1 x 10-4

Hexachlorobenzene 2.50 x 10-2 3.07 x 10-3 1 x 10-4

Lindane 3.07 x 10-2 3.78 x 10-3 1 x 10-4

Mirex 0.8 0.098 1 x 10-4

Toxaphene 3.63 x 10-2 4.46 x 10-3a 0.003

Chlorpyrifos 1.2 1.147 0.002

Diazinon 2.8 0.344 0.002

Disulfoton 0.16 0.019 0.002

Ethion 2 0.245 0.002

Terbufos 0.08 0.009 0.002

Oxyfluorfen 0.546 0.067 0.010

PAHs 5.47 x 10-3 6.73 x 10-4 1 x 10-6

PCBs (Total)

 Sum of Aroclorsa

 Non-ortho coplanar PCBs

 Other congeners/

 homologues

0.02 2.45 x 10-3 a 0.020

2 x 10-6

0.002

Dioxins/Furans 2.56 x 10-7 a 3.15 x 10-8 a 1 x 10-6

a Target analyte (total)s for which the analytical detection limit is likely to be at or above the

calculated SV depending on the analytical method selected. States must ensure that the

analytical method chosen provides detection limits lower than the selected SVs for all

25 target analytes for designation of green area waters.
APPENDIX B

B-7

recreational fishers and the state must define “unrestricted consumption” for the

consumer. For example, the state’s green areas may be defined as areas from

which fish consumers may safely eat four 8-ounce fish meals per month (or

approximately one fish meal per week) without any additional health risks. This

definition must be clearly communicated, particularly to members of high-end

fish-consuming groups such as some Native Americans, certain ethnic groups,

and subsistence fishers as well as to sensitive populations (pregnant women/

fetuses, nursing women and children). The state should clearly define for the

public both the meal size and meal frequency used in their green area designations so that high-end fish consumers do not erroneously assume that the

unrestricted consumption designation that is protective of recreational fishers

based on their consumption rate is also protective of subsistence fishers. In

addition, the state should provide the fish-consuming public with information on

the types of fish samples (whole fish, skin-on fillets, skin-off fillets, or other

sample types) used to establish the green area designation. Because skinning,

trimming, and certain cooking procedures also help reduce chemical residues in

fish tissues (EPA, 1999) (see Volume 2 of this series, Appendix C—Dose

Modification Due to Food Preparation), the state should also provide information

on these procedures particularly to fisher populations who consume whole fish

or portions of the fish other than the standard fillet. If the green area concept is

to be effective in promoting fishing and the consumption of fish, it is essential that

the fish-consuming public be given adequate information to understand the

definition of unrestricted fish consumption from these green areas.

One approach is to communicate these locations to the public in fishing

brochures annually distributed as part of the existing fish advisory programs. In

addition to publishing this information in state fishing brochures, EPA anticipates

making this information a new choice of advisory designations available to the

states and tribes for incorporation into the National Listing of Fish and Wildlife

Advisories (NLFWA) database. EPA realizes that this new designation will be

successful only if the states and tribes receive guidance and the information is

presented in an easily implemented format. 

In addition to implementation of green areas within their jurisdictions, states are

also encouraged to initiate or expand the use of general fish consumption

guidance for all fish. Several states provide advise on catching, cleaning,

cooking, and consumption of fish species. In some jurisdictions, states have

issued unlimited consumption or restricted consumption advisories for smaller

size classes of those species that are particularly popular with consumers. In this

way, the state is still encouraging the recreational aspects of fishing and

continued consumption of smaller-sized fish within a given species that typically

contain lower residues of chemical contaminants. As a result, the public is

encouraged to enjoy both the sport of fishing and the health benefits of eating

fish within the specific consumption guidance provided by the state. 
APPENDIX B

B-8

References

Burr, M.L., A.M. Fehily, J.F. Gilbert, S. Rogers, R.M. Holliday, P.M. Sweetnam,

P.C. Elwood, and N.M. Deadman. 1989. Effects of changes in fat, fish, and fibre

intakes on death and myocardial reinfarction: Diet and Reinfarction Trial (DART).

Lancet 2 (8666):757-761.

Datta,S., K. Ohyama, D.Y. Dunlap, and F. Matsumura. 1999. Evidence for

organochlone contamination in tissues of salmonids in Lake Tahoe. Ecotoxicol.

and Environ. Safety 42: 94-101. 

Dolecek, T.A., and G. Granditis. 1991. Dietary polyunsaturated fatty acids and

mortality in the Multiple Risk Factor Intervention Trial (MRFIT). World Rev. Nutr.

Diet 66:205-216.

Grieb, T.M., C.T. Driscoll, S.P. Gloss, C.L. Schofield, G.L. Bowie, and D.B.

Porcella. 1990. Factors affecting mercury accumulation in fish in the Upper

Michigan Peninsula. Environ. Toxicol. Chem. 9:919-930.

Henry, K.S., K. Kannan, B.W. Nagy, N.R. Kevern, M.J. Zabik, and J.P. Giesy.

1998. Concentrations and hazard assessment of organochlorine contaminants

and mercury in smallmouth bass from a remote lake in the Upper Peninsula of

Michigan. Arch. Environ. Contam Toxicol. 34:81-86.

Kimbrough, R.D. 1991. Consumption of fish: Benefits and perceived risk.

J. Toxicol. Environ. Health 33 (1):81-91.

Knapp, H.R., and G.A. Fitzgerald. 1989. The antihypertensive effects of fish oil.

A controlled study of polyunsaturated fatty acid supplements in essential

hypertension. N. Engl. J. Med. 320 (16):1037-1043.

Kromhout, D. 1993. Epidemiological aspects of fish in the diet. Proc. Nutr.

Soc. 52 (3):437-439.

Kromhout, D., E.B. Bosschieter, and C.dL. Coulander. 1985. The inverse relation

between fish consumption and 20-year mortality from coronary heart disease. N.

Engl. J. Med. 312 (19):1205-1209.

McVeigh, G. 1990. Arthritis and diet: a new look. Prevention 42 (10):40-45.

Norell, S.E., A. Ahlbom, M. Feychting, and N.L. Pedersen. 1986. Fish

consumption and mortality from coronary heart disease. Br Med. J. (Clin. Res.

Ed.) 293(6544):426.
APPENDIX B

B-9

Shekelle, R.B., L. Missell, O. Paul, A. M. Shryock, and J. Stamler. 1985. Fish

consumption and mortality from coronary heart disease (letter). N. Engl. J. Med.

313 (13):820

Simopoulos, A.P. 1991. Omega-3 fatty acids in health and disease and in growth

and development. Am. J. of Clin. Nutr. 54(3):438-463.

Sorensen, J.A., G.E. Glass, K.W. Schmidt, J.K. Huber, and G.R. Rapp Jr. 1990.

Airborne mercury deposition and watershed characteristics in relation to mercury

concentrations in water, sediments, plankton, and fish of eighty northern

Minnesota lakes. Environ. Sci. Technol. 24:1716-1727.

Swackhamer, D.L., and R.A. Hites. 1988. Occurrence and bioaccumulation of

organochlorine compounds in fishes from Siskiwit Lake, Isle Royale, Lake

Superior. Environ. Sci. Technol. 22:543-548. 

U.S. EPA (Environmental Protection Agency). 1998. Guidance for Conducting

Fish and Wildlife Consumption Surveys. EPA-823-B-98-007. Office of Water,

Washington, DC.

U.S. EPA (Environmental Protection Agency). 2000. Guidance for Assessing

Chemical Contaminant Data for Use in Fish Advisories—Risk Assessment and

Fish Consumption Limits. Volume 2. 3rd Edition, EPA-823-B-99-009. Office of

Water, Washington, DC.
APPENDIX C

USE OF INDIVIDUAL SAMPLES IN FISH

CONTAMINANT MONITORING PROGRAMS
APPENDIX C

C-3

APPENDIX C

USE OF INDIVIDUAL SAMPLES IN FISH

CONTAMINANT MONITORING PROGRAMS

The use of composite samples is often the most cost-effective method for estimating average tissue concentrations of analytes in target species populations to

assess chronic human health risks. However, there are some situations in which

individual sampling can be more appropriate from both ecological and risk

assessment perspectives. Individual sampling provides a direct measure of the

range and variability of contaminant levels in target fish populations. Information

on maximum contaminant concentrations in individual fish is useful in evaluating

acute human health risks. Estimates of the variability of contaminant levels

among individual fish can be used to ensure that studies meet desired statistical

objectives. For example, the population variance of a contaminant can be used

to estimate the sample size needed to detect statistically significant differences

in the mean contaminant concentration compared to the contaminant screening

values. Finally, the analysis of individual samples may be desirable, or

necessary, when the objective is to minimize the impacts of sampling on certain

vulnerable target populations, such as predators in headwater streams and

aquatic turtles, and in cases where the cost of collecting enough individuals for a

composite sample is excessive.

Analyzing individual fish incurs additional expenses, particularly when one

considers that a number of individual analyses are required to achieve measurements of a reasonable statistical power. However, the recommendation that

states archive the individual fish homogenates from which composite samples are

prepared for both screening and intensive studies (see Section 6.1.1.6) would

make it possible to perform individual analyses where needed without incurring

additional sampling costs.

Individual analysis is especially well-suited for intensive studies, in which results

from multiple stations and time periods are to be compared. The remainder of this

appendix discusses how the sampling design might be affected by analyzing

individual rather than composite samples and how contaminant data from

individuals versus composites might be used in risk assessments.

C.1 SAMPLING DESIGN

There are seven major components of the sampling design for a fish or shellfish

monitoring program: site selection, target species, target analytes, target analyte

screening values (SVs), sampling time, sampling type and size class, and repli-
APPENDIX C

C-4

Table C-1. Relative Cost of Obtaining Contaminant Data from

Individual Versus Composite Samples

Cost component

Relative cost

Composite samples Individual samples

Collection Moderate to high Low to moderate

Preparation Very low to moderate Very low to low

Analysis Low to moderate Moderate to high

cate samples. Of these, only the number of replicate samples and possibly the

target species would be expected to differ if individual samples were analyzed

rather than composites. Target species becomes a limiting factor when individuals of the target species are not large enough to provide adequate tissue mass

for all the required chemical analyses.

The five factors that determine the optimal number of fish or shellfish to analyze

are presented in Section 6.1.2.7. Briefly, the five factors are

• Cost components

• Minimum detectable difference between measured site-specific mean target

analyte concentration and SV

• Level of significance

• Population variance

• Power of the hypothesis test.

Each of these characteristics will be examined in detail for the collection and

analysis of individual samples.

C.1.1 Cost Components

The cost of obtaining contaminant data from individual fish or shellfish is

compared to the cost of obtaining contaminant data from composite samples in

Table C-1. These costs are dependent on the separate costs of collecting,

preparing, and analyzing the samples.

Typically, the cost of collecting individual samples will be less than that of

collecting composite samples when the target species is scarce or difficult to

capture. The cost of collecting individuals may not be a factor if the sample

collection method used typically allows for the collection of a large number of

individuals in a short period of time. In some situations, seines or gill nets might

have this characteristic. Also, in estuaries, coastal water, or large lakes where

productivity is high, the additional cost of collecting large numbers of individuals

for composite sampling may be minimal compared to the effort expended for collecting individual samples.
APPENDIX C

C-5

The cost of preparing individual samples for analysis is typically lower than either

the costs of collection or analysis. Generally, the cost of preparing composite

samples for analysis will be greater than that of preparing individual samples.

Sample preparation procedures can range in complexity from the grinding of

whole fish to delicate and time-consuming operations to resect specific tissues.

Costs of composite sampling depend largely on the number of individuals required

per composite sample and the number of replicate composite samples required

to achieve the desired statistical power; however, these costs can be somewhat

controlled (see Section 6.1.2.7).

The cost of analyzing individual samples is also typically higher than the cost of

analyzing composite samples. The cost differential between the two approaches

is directly correlated to the cost for the analysis of a single sample. For some

intensive studies, the number of target analytes exceeding the SV is small, so few

analyses are required. In these cases, the relative costs between the two

approaches may not differ greatly if the number of samples analyzed using the

two different approaches is similar (e.g., three to five samples). A sampling

design with such a small number of individual samples would be appropriate only

if the expected mean target analyte concentration was much greater than the SV.

C.1.2 Minimum Detectable Difference

The difference between the mean target analyte concentration at a site and the

SV will not often be known before the screening study has been performed. The

minimum detectable difference between the mean concentration and the SV will

depend on the level of significance (see Section C.1.3), population variance

(Section C.1.4), and the number of replicates collected. In practice, the sample

size is often determined by establishing the minimum detectable difference prior

to the study according to the objectives of the project. For an SV that has not

been multiplied by an uncertainty factor, the cost of detecting a 10 percent

difference may be warranted. The issue of minimum detectable difference is

discussed in greater detail in Section C.1.5.

C.1.3 Level of Significance

The level of significance (LS) refers to the probability of incorrectly rejecting the

null hypothesis that there is no difference between the mean target analyte concentration and the SV. This probability is also called Type I error. The LS can be

thought of as the chance of a "false positive" or of detecting a difference that does

not exist. The LS affects the sampling design by modifying the required power

(thus impacting the sample size) of the statistical test to detect a significant

difference between the mean target analyte concentration and the SV (see

Section C.1.5). A typical LS used in biological sampling is 0.05. In some cases,

an LS other than 0.05 could be appropriate. If the ramifications of a statistically

significant difference are severe, a more conservative LS (e.g., 0.01) might be

used. On the other hand, if the statistical test is being conducted to identify
APPENDIX C

C-6

n=

(Z +Z a b s

d

)

2 2

2

2

whether additional sampling should be performed (i.e., a screening survey), then

a less conservative LS (e.g., 0.10) might be used.

C.1.4 Population Variance

The variability in target analyte concentrations within a given fish or shellfish

population is a critical factor in determining how many individual samples to collect

and analyze. The population variance directly affects the power of the statistical

test to detect a significant difference between the mean target analyte

concentration and the SV (see Section C.1.5) by impacting the sample size. The

population variance may not be known prior to sampling, but it can be estimated

from similar data sets from the same target species, which could in many cases

be obtained by analyzing individual fish homogenates if these have been archived

as recommended in Section 6.1.1.6. In using historical data to estimate

population variance, it is important to consider contaminant data only from

individual fish or shellfish of the same species. By its very nature, a data set

consisting of replicate composite samples tends to smooth out the variability

inherent in a group of individual organisms. An extreme example of this

phenomenon was presented by Fabrizio et al. (1995) in a study on procedures for

compositing fish samples. They used computer simulations to predict PCB

concentrations in composite samples of striped bass that had previously been

analyzed individually. The predicted variance in these concentrations in the

composite samples was approximately 20 percent of the variance obtained from

individual analyses.

C.1.5 Power of Statistical Test

Another critical factor in determining the sample size is the power of the statistical

test, that is, the probability of detecting a true difference between the mean target

analyte concentration and the SV. Because of its profound influence on sample

size, it is the power of the test that may ultimately control whether the objectives

of the survey are met. The effect of joint consideration of the desired power, the

population variance, and the minimum detectable difference on the sample size

is described by the following formula (Steel and Torrie, 1980):

where

n = sample size

Z" = Z statistic for Type I error ()

Z$ = Z statistic for Type II error ()

2 = population variance (estimated from historical data)

 = minimum detectable difference between mean target analyte

concentration and SV.
APPENDIX C

C-7

Recall that the Type I error is equal to the LS, and the value is generally between

0.01 and 0.10. Type II error is the probability of accepting the null hypothesis

(that there is no difference between the mean target population concentration and

the SV) when it is actually false. This type of error can be thought of as the

chance of a "false negative," or not detecting a difference that does in fact exist.

The complement of Type II error (1-) is the power of the statistical test.

The above equation for determining sample size was solved for powers ranging

from 0.5 to 0.9 (50 to 90 percent; Figure C-1) assuming an LS of 0.05. The

values for  (standard deviation) and  were set relative to the SV. A similar

exercise was performed in Section 6.1.2.7 and two examples were provided. In

example A, both the standard deviation and minimum detectable difference were

set to 0.5 SV. Example A corresponds to a ratio of 1 on the x-axis of Figure C-1.

Applying example A to the collection of individual fish, the recommended sample

size would range from approximately 6 individual samples for a power of 50

percent to 18 individual samples for a power of 90 percent (Figure C-1). In

example B, the standard deviation was set to 1.0 SV, while the minimum

detectable difference was kept at 0.5 SV. Example B corresponds to a ratio of 2

on the x-axis of Figure C-1. Applying example B to the collection of individual

samples, the sample size would have to be almost 40 individual samples to

achieve even a modest statistical power (i.e., 70 percent).

It is common to set the power of the statistical test to at least 80 percent

(Fairweather, 1991). Figure C-1 indicates that, to achieve a statistical power of

80 percent using the variability assumptions in examples A and B, 13 and 50 fish

would have to be collected, respectively. The estimated sample sizes for

individual fish or shellfish is similar to those calculated for composite samples (see

Section 6.1.2.7). For example A as applied to composite samples, 12 to 18 fish

would have to be collected. For example B as applied to composite samples, 30

to 50 fish would have to be collected. Thus, the cost of collecting the fish to

achieve a power of 80 percent would not be significantly different for composite

versus individual samples (see Section C.1.1). The number of analyses, however,

would be considerably less for composite samples (3 to 10 analyses of composite

samples versus 13 or 50 analyses of individual samples).

Figure C-1 also indicates that 10 or fewer individual fish or shellfish should be

analyzed only if the ratio of the standard deviation to the minimum detectable

difference is 0.85 or less. For ratios less than 0.5, the effect of sample size on the

statistical power is minor. If the expected mean target analyte concentration is

many times greater than the SV, it may not be necessary to allocate resources

toward the collection and analysis of more than a minimum number (e.g., three

to five samples) of individual fish or shellfish.
C-8Figure C-1. Recommended sample sizes to achieve various statistical powers.
APPENDIX C

C-9

C.2 USE OF CONTAMINANT DATA FROM INDIVIDUAL FISH/SHELLFISH IN RISK

ASSESSMENTS

Target analyte concentrations in composite samples represent averages for

specific target species populations. The use of these values in risk assessments

is appropriate if the objective is to estimate the average concentration to which

consumers of the target species might be exposed over a long period of time.

The use of long exposure durations (e.g., 30 to 70 years) is typical of the

assessment of carcinogenic target analytes, the health effects of which may be

manifested over an entire lifetime (see Volume 2 of this series). Target analytes

that produce noncarcinogenic effects, on the other hand, may cause acute effects

to human health over a relatively short period of time on the order of hours or

days. The use of average contaminant concentrations derived from the analysis

of composite samples may not be protective against acute health effects because

high concentrations in an individual organism may be masked by lower

concentrations in other individuals in the composite sample. Contaminant data

from individual samples permits the use of alternative estimates of contaminant

concentration for a group of fish or shellfish (e.g., maximum). Therefore, the

decision whether to collect and analyze individual fish or shellfish may depend on

the target analytes included in the monitoring program.

EPA has recommended that 25 target analytes be included in screening studies

(see Section 4). All of the target analytes except PCBs, PAHs, and dioxins/furans

have reference doses for noncarcinogenic health effects, although the

carcinogenic risk is likely to be greater than the noncarcinogenic risk for eight

other target analytes (see Tables 5-2 and 5-3). EPA’s reassessment of the health

effects of 2,3,7,8-TCDD (dioxin) indicated that this chemical may also pose a

significant noncarcinogenic health risk in some cases (U.S. EPA, 1994).

C.3 EXAMPLE CASE STUDY

The presentation of a case study will illustrate some of the sample size and data

interpretation issues discussed in Sections C.1 and C.2, respectively. A State has

prepared a composite sample of target species A from a particular waterbody of

concern. This composite sample was analyzed for all 25 target analytes listed in

Table 4-1. Of the 25 target analytes, only cadmium was detected at a

concentration exceeding the state selected SV (10 ppm) for cadmium. Cadmium

was detected at 20 ppm, twice the SV calculated for cadmium. Because the SV

for at least one target analyte was exceeded, an intensive study was warranted.

The state decided to collect and analyze individual fish in the intensive study for

the following reasons: (1) the cost of collecting individual fish is less than the cost

of collecting fish for composites, (2) the analytical costs for analyzing cadmium

are relatively low (<$50 sample), and (3) the cadmium concentrations in individual

fish should more accurately reflect the potential acute (noncarcinogenic) health

risk from cadmium than the mean cadmium concentration derived from composite

samples.
APPENDIX C

C-10

The first issue the state must decide is how many individual fish to collect and

analyze. The important factors in this decision are the minimum detectable

difference the state wishes to test and the variability in cadmium concentrations

within the target species population. The first factor can be obtained from the

results of the screening survey. The state wishes to test whether the difference

between the concentration detected in the single composite sample (20 ppm) and

the selected SV (10 ppm) is significant. This assumes that the mean cadmium

concentration for the individual is also 20 ppm. The expected standard deviation

(8 ppm) was obtained from a previous investigation performed on individuals of

the target species and was equal to 0.8 of the SV (10 ppm). Using Figure C-1,

it can be seen that, for a ratio of standard deviation (0.8 x SV) to detectable

difference (1.0 x SV) of 0.8, the sample size necessary to achieve a statistical

power of 80 percent would be eight fish.

The state determines that the mean cadmium concentration of eight individual fish

of the target species is 30 ppm and the standard deviation is equal to the

predicted value of 8 ppm. The state performs a t-test to determine if the mean

concentration is significantly greater than the SV. As described in Section 6.1.2.7,

the statistic

(mean - SV)/standard deviation

has a t-distribution with n-1 degrees of freedom. For this example, the t statistic

is 2.5 ([(30-10)/8] with 7 degrees of freedom. This value exceeds the critical

t-statistic (1.895) for a one-tailed LS of 0.05. Therefore, the state determines that

the mean cadmium concentration for these eight individual fish of the target

species is significantly greater than the SV and a risk assessment is performed.

C.4 REFERENCES

Fabrizio, M.C., A.M. Frank, and J.F. Savino. 1995. Procedures for formation of

composite samples from segmented populations. Environmental Science

and Technology 29(5):1137-1144.

Fairweather, P.G. 1991. Statistical power and design requirements for environmental monitoring. Aust. J. Freshwater Res. 42:555-567.

Steel, R.G.D., and J.H. Torrie. 1980. Principles and Procedures of Statistics. A

Biometrical Approach. Second Edition. McGraw-Hill Book Company.

New York, NY. 633 pp.

U.S. EPA (U.S. Environmental Protection Agency). 1994. Health Assessment for

2,3,7,8-TCDD and Related Compounds. Public Review Draft.

EPA/600/EP-92/001.
APPENDIX D

FISH AND SHELLFISH SPECIES FOR WHICH STATE

CONSUMPTION ADVISORIES HAVE BEEN ISSUED
APPENDIX D

D-3

APPENDIX D

FISH AND SHELLFISH SPECIES FOR WHICH STATE CONSUMPTION

ADVISORIES HAVE BEEN ISSUED

FRESHWATER FINFISH SPECIES FOR WHICH STATE

CONSUMPTION ADVISORIES HAVE BEEN ISSUED 

1993 and 1998 (Bold type) 

1998 only (Normal type)

1993 only (Italic type)

AL fish species (unspecified), catfish (unspecified), bigmouth buffalo,

brown bullhead, channel catfish, white bass

smallmouth buffalo, channel catfish, largemouth bass, spotted bass, striped

bass, crappie, king mackerel

AK no consumption advisories

AS fish species (unspecified), shellfish (unspecified)

AZ fish species (unspecified)

largemouth bass, yellow bullhead, channel catfish, black crappie, bluegill

sunfish, green sunfish, redear sunfish

AR fish species (unspecified)

bass (unspecified), black bass, largemouth bass, bowfin, buffalo, catfish

(unspecified), blue catfish, channel catfish, flathead catfish, crappie,

freshwater drum, gar, pickerel, redhorse, sucker, sunfish

CA goldfish, Sacramento blackfish, brown bullhead, crappie

(unspecified), hitch, largemouth bass, smallmouth bass, channel

catfish, white catfish, trout (unspecified), rainbow trout, croaker

(unspecified), orangemouth corvina, sargo, tilapia (unspecified), fish

species (unspecified)

brown trout, bullhead (unspecified), white crappie, black crappie, carp

(unspecified), corbina, striped bass, kelp bass, gobies, queenfish, rockfish,

sculpin, shark, shellfish (unspecified), surfperch

squawfish, sucker (unspecified)
APPENDIX D

D-4

CO rainbow trout, yellow perch, northern pike, walleye, smallmouth bass,

largemouth bass, black crappie, kokanee salmon, channel catfish, fish

species (unspecified)

 bullhead, common carp, crappie (unspecified), brown trout

trout (unspecified)

CT common carp, fish species (unspecified)

 largemouth bass, smallmouth bass, striped bass, bluefish, bullhead, catfish

(unspecified), American eel, chain pickerel, trout (unspecified)

DE white catfish, channel catfish, fish species (unspecified)

striped bass, white perch, carp (unspecified), largemouth bass, catfish

(unspecified)

DC American eel

carp (unspecified), catfish (unspecified)

channel catfish, common carp

FL largemouth bass, gar, bowfin, warmouth sunfish, yellow bullhead,

Mayan cichlid, oscar, spotted sunfish

gafftopsail catfish, jack crevalle, ladyfish, Spanish mackerel, king mackerel,

spotted sea trout, shark

GA common carp, largemouth bass, catfish (unspecified), fish species

(unspecified)

hybrid bass, redeye bass, shoal bass, spotted bass, striped bass,

Suwannee bass, white bass, bowfin, smallmouth buffalo, bullhead

(unspecified), brown bullhead, spotted bullhead, yellow bullhead, channel

catfish, flathead catfish, white catfish, black crappie, Atlantic croaker, black

drum, red drum, flounder, striped mullet, silver perch, chain pickerel, grayfin

redhorse, silver redhorse, spotted sea trout, greater jumprock sucker,

spotted sucker, sunfish, redbreast sunfish, rainbow trout, walleye, clams,

blue crab, mussels, oysters, shrimp

GU no consumption advisories

HI all fish species (unspecified), shellfish (unspecified)

ID smallmouth bass, catfish (unspecified), black crappie, white crappie, yellow

perch

IL lake trout, coho salmon, chinook salmon, brown trout, common carp,

catfish (unspecified), channel catfish, shovelnose sturgeon, bluegill,

freshwater drum, largemouth bass, spotted bass, alewife

 white bass, blue catfish, black crappie, white crappie, yellow perch, sauger,

smelt

bigmouth buffalo, flathead catfish, smallmouth buffalo, crappie (unspecified)
APPENDIX D

D-5

IN fish species (unspecified), common carp, catfish (unspecified), coho

salmon, brown trout, lake trout, chinook salmon, channel catfish

largemouth bass, rock bass, smallmouth bass, spotted bass, striped bass,

hybrid striped bass, white bass, yellow bass, bloater, bowfin, bigmouth

buffalo, black buffalo, smallmouth buffalo, bullhead (unspecified), black

bullhead, yellow bullhead, carp (unspecified), carpsucker, quillback

carpsucker, river carpsucker, flathead catfish, creek chub, black crappie,

white crappie, freshwater drum, round goby, northern hogsucker,

paddlefish, yellow perch, northern pike, redhorse (unspecified), black

redhorse, golden redhorse, river redhorse, shorthead redhorse, silver

redhorse, sauger, gizzard shad, shovelnose sturgeon, blue sucker,

longnose sucker, spotted sucker, white sucker, bluegill sunfish, green

sunfish, longear sunfish, brook trout, rainbow trout, steelhead trout,

walleye, whitefish (unspecified), lake whitefish

IA common carp, fish species (unspecified)

quillback carpsucker

channel catfish, carpsucker (unspecified)

KS buffalo (unspecified), common carp, freshwater drum, carpsucker

(unspecified)

blue catfish, channel catfish, flathead catfish, bullhead catfish, shovelnose

sturgeon

catfish (unspecified), sturgeon (unspecified)

KY channel catfish, paddlefish, white bass, common carp, fish species

(unspecified)

largemouth bass

LA bass (unspecified), fish species (unspecified)

largemouth bass, spotted bass, striped bass, white bass, bowfin, bigmouth

buffalo, carp (unspecified), channel catfish, flathead catfish, crappie, black

crappie, white crappie, freshwater drum, gar, king mackerel, shad, shellfish

(unspecified), bluegill sunfish, redear sunfish

ME fish species (unspecified)

striped bass, bluefish, American lobster,l freshwater fish, cold water fish,

warm water fish

MD channel catfish, American eel, black crappie, common carp

bullhead (unspecified), sunfish (unspecified)
APPENDIX D

D-6

MA brown trout, yellow perch, white sucker, American eel, smallmouth

bass, largemouth bass, lake trout, channel catfish, brown bullhead,

common carp, white catfish, fish species (unspecified)

bottom fish, bass (unspecified), yellow bullhead, black crappie, flounder,

white perch, scup, chain pickerel, bivalves (unspecified), lobster

(unspecified), American lobster, tautog

MI common carp, rock bass, yellow perch, largemouth bass, smallmouth

bass, walleye, northern pike, muskellunge, white bass, longnose

sucker, white perch, brown bullhead, bullhead (unspecified), bluegill,

brown trout, siscowet trout, lake trout, coho salmon, chinook salmon,

splake, catfish (unspecified), rainbow trout, sucker (unspecified),

gizzard shad, freshwater drum, white sucker, lake whitefish

fish species (unspecified), yellow bullhead, burbot, quillback carpsucker,

channel catfish, black crappie, white crappie, redhorse, smelt, lake sturgeon

crappie (unspecified), sauger, carpsucker (unspecified), sturgeon

(unspecified), brook trout

MN yellow perch, brown bullhead, black bullhead, yellow bullhead,

quillback carpsucker, brown trout, brook trout, lake trout, chinook

salmon, ciscowet, walleye, northern pike, muskellunge, splake, smallmouth bass, largemouth bass, rock bass, white bass, rainbow trout,

white sucker, bluegill, black crappie, white crappie, shorthead

redhorse, silver redhorse, common carp, smallmouth buffalo, sauger,

bigmouth buffalo, channel catfish, lake whitefish, freshwater drum,

pumpkinseed, lake herring, flathead catfish, bowfin, siscowet trout 

bass (unspecified), burbot, carp (unspecified), cisco, crappie (unspecified),

redhorse, golden redhorse, coho salmon, lake sturgeon

tullibee, redhorse sucker, chub bloater

MS fish species (unspecified), catfish (unspecified)

largemouth bass, spotted bass, king mackerel

buffalo (unspecified)

MO sturgeon (unspecified),, buffalo (unspecified), sucker (unspecified),

paddlefish, catfish (unspecified), redhorse, freshwater drum

carp (unspecified), fish species (unspecified), sunfish 

common carp, channel catfish, flathead catfish

MT fish species (unspecified), largemouth bass, smallmouth bass, burbot,

black crappie, yellow perch, northern pike, kokanee salmon, white sucker,

brook trout, brown trout, bull trout, cutthroat trout, lake trout, rainbow trout,

walleye, lake whitefish, mountain whitefish
APPENDIX D

D-7

NE common carp, channel catfish

largemouth bass, catfish (unspecified), northern pike, fish species

(unspecified)

NV fish species (unspecified)

NH freshwater fish (unspecified), largemouth bass, bluefish, American lobster

fish species (unspecified)

NJ striped bass, American eel, white perch, white catfish, fish species

(unspecified)

largemouth bass, bluefish, chain pickerel, blue crab, crustaceans

(unspecified), American lobster, molluscs (unspecified)

NM white crappie, channel catfish, common carp, brown trout, river

carpsucker, kokanee salmon, largemouth bass, bluegill, white bass,

white sucker, yellow perch, black bullhead, black crappie, bass

(unspecified), crappie (unspecified), rainbow trout, longnose dace,

walleye, northern pike, bullhead (unspecified), black bass

smallmouth bass, spotted bass

trout (unspecified), carpsucker (unspecified)

NY common carp, lake trout, brown trout, yellow perch, smallmouth bass,

splake, American eel, goldfish, striped bass, white perch, bluefish,

largemouth bass, brown bullhead, white catfish, walleye, rainbow

smelt, tiger muskellunge, white sucker, chinook salmon, coho salmon,

rainbow trout

fish species (unspecified), sportfish (unspecified), channel catfish, Atlantic

needlefish, blue crab, American lobster, brook trout

northern pike

NC largemouth bass, fish species (unspecified)

bowfin, common carp, catfish (unspecified), white catfish, black crappie,

white crappie, green sunfish, redear sunfish 

ND walleye, white bass, yellow perch, northern pike, bigmouth buffalo,

common carp, crappie (unspecified), white sucker, channel catfish,

goldeye, sauger, smallmouth bass

largemouth bass, brown bullhead, black crappie, white crappie, paddlefish,

bluegill, brown trout, rainbow trout

bullhead (unspecified), chinook salmon, carpsucker (unspecified), sunfish

(unspecified)
APPENDIX D

D-8

OH common carp, catfish (unspecified), white bass, sucker (unspecified),

fish species (unspecified)

largemouth bass, rock bass, smallmouth bass, spotted bass, hybrid striped

bass, brown bullhead, yellow bullhead, channel catfish, flathead catfish,

white crappie, freshwater drum, white perch, chinook salmon, coho salmon,

sauger, white sucker, lake trout, steelhead trout, walleye

OK largemouth bass

catfish (unspecified)

channel catfish, fish species (unspecified)

OR fish species (unspecified), crayfish

largescale sucker, brown trout, black crappie, squawfish, largemouth bass,

smallmouth bass, common carp, peamouth chub

PA white sucker, white perch, common carp, American eel, channel

catfish, goldfish, largemouth bass, quillback carpsucker, white bass,

lake trout, walleye, smallmouth bass, shorthead redhorse, sucker

(unspecified), fish species (unspecified)

spotted bass, hybrid striped bass, bowfin, flathead catfish, crappie,

freshwater drum, muskellunge, northern pike, coho salmon, sauger,

sunfish, bluegill, brown trout, rainbow trout, lake whitefish

green sunfish

PR no fish consumption advisories

RI striped bass

fish species (unspecified), bluefish

SC fish species (unspecified)

largemouth bass, bowfin, catfish (unspecified), channel catfish, bluegill

sunfish, redear sunfish

shellfish (unspecified)

SD no fish consumption advisories

TN catfish (unspecified), largemouth bass, common carp, striped bass,

sauger, white bass, smallmouth buffalo, fish species (unspecified)

hybrid striped bass, redbreast sunfish

crappie (unspecified), rainbow trout

TX catfish (unspecified), fish species (unspecified)

largemouth bass, hybrid striped bass, white bass, freshwater drum, king

mackerel, crab (unspecified), blue crab
APPENDIX D

D-9

UT fish species (unspecified)

VT brown trout, lake trout

fish species (unspecified)

walleye

VA fish species (unspecified)

smallmouth bass, striped bass, white bass, common carp, channel catfish,

flathead catfish

VI no fish consumption advisories

WA bottomfish species (unspecified), common carp, rockfish, shellfish

(unspecified), crab (unspecified), sucker, bridgelip sucker, lake whitefish,

mountain whitefish

no fish consumption advisories in 1993

WV channel catfish, common carp, sucker (unspecified), fish species

(unspecified)

bottomfish species (unspecified), non-sportfish (unspecified), largemouth

bass, smallmouth bass, hybrid striped bass, white bass, flathead catfish,

freshwater drum, sauger

brown bullhead

WI coho salmon, chinook salmon, common carp, catfish (unspecified),

splake, rainbow trout, lake trout, brown trout, siscowet trout, northern

pike, white bass, white sucker, walleye, yellow perch, muskellunge,

flathead catfish, freshwater drum, channel catfish, bullhead

(unspecified), bluegill, black crappie, crappie (unspecified), rock bass,

smallmouth bass, redhorse (unspecified), largemouth bass, lake

sturgeon, buffalo (unspecified), fish species (unspecified)

bigmouth buffalo, brown bullhead, carp (unspecified), chub, lake herring,

white perch, northern redhorse, sauger, sheepshead, smelt, green sunfish,

pumpkinseed, lake whitefish, panfish species (unspecified)

brook trout

WY no fish consumption advisories
APPENDIX D

D-10

ESTUARINE/MARINE FISH AND SHELLFISH SPECIES FOR WHICH STATE

CONSUMPTION ADVISORIES HAVE BEEN ISSUED

1993 and 1998 (Bold type) 

1998 only (Normal type)

1993 only (Italic type)

AL king mackerel

no consumption advisories in 1993

AK no consumption advisories

AS fish and shellfish species (unspecified)

CA white croaker, black croaker, corbina, surfperch, queenfish, sculpin,

rockfish, kelp bass, striped bass, fish and shellfish species

(unspecified)

bullhead, croaker (unspecified), gobies, shark

CT striped bass, bluefish

DE fish species (unspecified), striped bass, channel catfish, white catfish, white

perch, carp, catfish (unspecified), largemouth bass

no consumption advisories in 1993

DC no consumption advisories in 1998

channel catfish, American eel 

FL shark (unspecified)

 gafftopsail catfish, crevalle jack, ladyfish, king mackerel, Spanish mackerel,

spotted sea trout, yellow bullhead, gar, warmouth sunfish, bowfin

GA Atlantic croaker, black drum, red drum, flounder, silver perch, spotted sea

trout, clams, blue crab, mussels, oysters

no consumption advisories in 1993

GU no consumption advisories

HI fish and shellfish species (unspecified)

no consumption advisories in 1993

LA fish and shellfish species (unspecified)

 king mackerel

ME striped bass, bluefish, American lobster,

no consumption advisories in 1993

MD channel catfish, American eel
APPENDIX D

D-11

MA American eel, flounder, American lobster, bivalves (unspecified), fish

species (unspecified)

 bottomfish (unspecified), scup, tautog

MS king mackerel

no consumption advisories in 1993

NH bluefish, American lobster

no consumption advisories in 1993

NJ striped bass, bluefish, American eel, white perch, white catfish, blue

crab

American lobster

fish and shellfish species (unspecified)

NY American eel, striped bass, bluefish, white perch, white catfish,

rainbow smelt, Atlantic needlefish, blue crab

American lobster, fish species (unspecified), largemouth bass, smallmouth

bass, common carp, goldfish, walleye

NC fish species except herring, shad, striped bass, and shellfish species

(unspecified)

OR no consumption advisories

PA white perch, channel catfish, American eel

PR no consumption advisories

RI striped bass, bluefish

SC bluegill, bowfin, largemouth bass

fish and shellfish species (unspecified)

TX blue crab, catfish (unspecified), fish species (unspecified)

crab (unspecified), king mackerel

VA no consumption advisories

fish species (unspecified)

VI no consumption advisories

WA bottomfish species (unspecified), rockfish, shellfish (unspecified), crab

(unspecified)

no consumption advisories in 1993
APPENDIX D

D-12

SOURCES

RTI (Research Triangle Institute). 1993. National Listing of State Fish and

Shellfish Consumption Advisories and Bans. (Current as of July 22, 1993.)

Research Triangle Park, NC.

U.S. EPA (Environmental Protection Agency). 1999. National Listing of Fish and

Wildlife Consumption Advisories Database. Office of Water. Washington, DC.
APPENDIX E

TARGET ANALYTES ANALYZED IN NATIONAL OR

REGIONAL MONITORING PROGRAMS
APPENDIX E

(continued) 

E-3

Table E-1. Target Analytes Analyzed in National or Regional Monitoring 

Programs Reviewed by the 1993 Fish Contaminant Workgroup

Monitoring program

Analyte a b c1 de fgh

Metals

 Aluminum (Al)  

 Antimony (Sb)  

 Arsenic (As) (total)    

 Barium (Ba) 

 Beryllium (Be)  

 Cadmium (Cd)    

 Chromium (Cr)   

 Copper (Cu)    

 Cyanide 

 Iron (Fe)  

 Lead (Pb)    

 Manganese (Mn)  

 Mercury (Hg)   

 Methylmercury 

 Molybdenum 

 Nickel (Ni)  

 Selenium (Se)  

 Silicon (Si) 

 Silver (Ag)  

 Thallium (Tl)  

 Tin (Sn) 

 Tributyltin

 Vanadium 

 Zinc (Zn)  

Pesticides

 Aldrin   

 Butachlor 

 Chlordane (cis & trans)   2 

 Chlorpyrifos 

 Danitol 
Table E-1. (continued)

APPENDIX E

Monitoring program

Analyte a b c1 de fgh

(continued) 

E-4

 DCPA (chlorthal) 

 DDT (total)  

2,4’-DDD (2,4’-TDE)   

4,4’-DDD (4,4’-TDE)   

2,4’-DDE   

4,4’-DDE  

2,4’-DDT   

4,4’-DDT   

 Demeton 

 Dicofol  

 Dieldrin  

 Diphenyl disulfide 

 Endosulfan

-Endosulfan (endosulfan I) 

ß-Endosulfan (endosulfan II) 

Endosulfan sulfate 

 Endrin   

 Endrin aldehyde 

 Ethyl-p-nitrophenylphenylphosphorothioate (EPN) 

 Fonofos

 Guthion 

 Heptachlor  

 Heptachlor epoxide   

 Hexachlorocyclohexane (HCH) also known

 as Benzene hexachloride (BHC)

-Hexachlorocyclohexane  

ß-Hexachlorocyclohexane   

-Hexachlorocyclohexane  

-Hexachlorocyclohexane (lindane)  

Technical-hexachlorocyclohexane 

 Hexachlorophene 

 Isopropalin  

 Kepone 
Table E-1. (continued)

APPENDIX E

Monitoring program

Analyte a b c1 de fgh

(continued) 

E-5

 Malathion 

 Methoxychlor  

 Mirex  

 Nitrofen 

 cis-Nonachlor  

 trans-Nonachlor 

 Oxychlordane  

 Parathion 

 Toxaphene (mixture)   

 Triazine herbicides

 Trichloronate 

 Trifluralin  

Base/Neutral Organic Compounds

 Acenaphthene  

 Acenaphthylene  

 Anthracene  

 Benzidine 

 Benzo(a)anthracene  

 Benzo(a)pyrene  

 Benzo(e)pyrene 

 Benzo(b)fluoranthene  

 Benzo(k)fluoranthene  

 Benzo(g,h,i)perylene  

 Benzyl butyl phthalate 

 Biphenyl  

 4-Bromophenyl ether 

 bis(2-Chloroethoxy)methane 

 bis(2-Chloroethyl)ether 

 bis(2-Chloroisopropyl)ether 

 bis(2-Ethylhexyl)phthalate (BEHP) 

 Chlorinated benzenes

 2-Chloronaphthalene 
Table E-1. (continued)

APPENDIX E

Monitoring program

Analyte a b c1 de fgh

(continued) 

E-6

 4-Chlorophenyl ether 

 Chrysene  

 Dibenzo(a,h)anthracene  

 Di-n-butyl phthalate 

 1,2-Dichlorobenzene 

 1,3-Dichlorobenzene 

 1,4-Dichlorobenzene 

 3,3’-Dichlorobenzidine  

 Diethyl phthalate 

 2,6-Dimethylnaphthalene  

 2,3,5-Trimethylnaphthalene 

 Dimethyl phthalate 

 2,4-Dinitrotoluene 

 2,6-Dinitrotoluene 

 Di-n-octyl phthalate 

 1,2-Diphenylhydrazine 

 bis(2-Ethylhexyl) phthalate 

 Fluoranthene  

 Fluorene  

 Heptachlorostyrene 

 Hexachlorostyrene 

 Hexachlorobenzene  

 Hexachlorobutadiene  

 Hexachlorocyclopentadiene  

 Hexachloroethane 

 Indeno(1,2,3-cd)pyrene  

 Isophorone 

 4,4’-Methylene bis(N,N’-dimethyl)aniline 

 1-Methylnaphthalene 

 2-Methylnaphthalene 

 1-Methylphenanthrene 

 Naphthalene  
Table E-1. (continued)

APPENDIX E

Monitoring program

Analyte a b c1 de fgh

(continued) 

E-7

 Nitrobenzene 

 N-Nitroso-di-n-butylamine 

 N-Nitrosodimethylamine 

 N-Nitrosodiphenylamine 

 N-Nitrosodipropylamine 

 Octachlorostyrene  

 PAHs (polycyclic aromatic hydrocarbons)

 PBBs (polybrominated biphenyls) 

 PCBs (polychlorinated biphenyls)  

Aroclor 1016 (mixture)  

Aroclor 1221 (mixture)  

Aroclor 1232 (mixture)  

Aroclor 1242 (mixture)  

Aroclor 1248 (mixture)  

Aroclor 1254 (mixture)  

Aroclor 1260 (mixture)  

Selected individual congeners 

 Pentachloroanisole (PCA)  

 Pentachlorobenzene  

 Pentachloronitrobenzene (PCNB)  

 Pentachlorophenyl methyl ether

 Pentachlorophenyl methyl sulfide

 Pentachlorostyrene 

 Perthane  

 Perylene 

 Phenanthrene  

 Pyrene  

 Terphenyl 

 1,2,3,4-Tetrachlorobenzene  

 1,2,3,5-Tetrachlorobenzene  

 1,2,4,5-Tetrachlorobenzene  

 1,2,3-Trichlorobenzene 
Table E-1. (continued)

APPENDIX E

Monitoring program

Analyte a b c1 de fgh

(continued) 

E-8

 1,2,4-Trichlorobenzene  

 1,3,5-Trichlorobenzene  

Triphenyl phosphate 

Dioxins

 1,2,3,7,8-Pentachlorodibenzodioxin (PeCDD)  

 2,3,7,8-Tetrachlorodibenzodioxin (TCDD)   

 1,2,3,4,6,7,8-Heptachlorodibenzodioxin (HpCDD)  

 1,2,3,4,7,8-Hexachlorodibenzodioxin (HxCDD)  

 1,2,3,6,7,8-Hexachlorodibenzodioxin (HxCDD)  

 1,2,3,7,8,9-Hexachlorodibenzodioxin (HxCDD)  

Dibenzofurans

 1,2,3,4,6,7,8-Heptachlorodibenzofuran (HpCDF)  

 1,2,3,4,7,8,9-Heptachlorodibenzofuran (HpCDF)  

 1,2,3,4,7,8-Hexachlorodibenzofuran (HxCDF)  

 1,2,3,6,7,8-Hexachlorodibenzofuran (HxCDF)  

 1,2,3,7,8,9-Hexachlorodibenzofuran (HxCDF)  

 2,3,4,6,7,8-Hexachlorodibenzofuran (HxCDF)  

 1,2,3,7,8-Pentachlorodibenzofuran (PeCDF)  

 2,3,4,7,8-Pentachlorodibenzofuran (PeCDF)  

 2,3,7,8-Tetrachlorodibenzofuran (TCDF)  

Acidic Organic Compounds

 Chlorinated phenols

 4-Chloro-3-cresol 

 2-Chlorophenol 

 2,4-Dichlorophenol 

 2,4-Dimethylphenol 

 4,6-Dinitro-2-cresol 

 2-4-Dinitrophenol 

 2-Nitrophenol 

 4-Nitrophenol 

 Pentachlorophenol (PCP)  
Table E-1. (continued)

APPENDIX E

Monitoring program

Analyte a b c1 de fgh

(continued) 

E-9

 Phenol 

 2,4,6-Trichlorophenol 

Volatile Organic Compounds

 Acrolein 

 Acrylonitrile 

 Benzene 

 Bromodichloromethane 

 Bromoform 

 Bromomethane 

 Carbon tetrachloride 

 Chlorobenzene 

 Chloroethane 

 2-Chloroethylvinyl ether 

 Chloroform 

 Chloromethane 

 Dibromochloromethane 

 1,1-Dichloroethane 

 1,2-Dichloroethane 

 1,1-Dichloroethene 

 trans-1,2-Dichloroethene 

 1,2-Dichloropropane 

 cis-1,3-Dichloropropene 

 trans-1,3-Dichloropropene 

 Ethylbenzene 

 Methylene chloride 

 1,1,2,2-Tetrachloroethane 

 Tetrachloroethene 

 Toluene 

 1,1,1-Trichloroethane 

 1,1,2-Trichloroethane 

 Trichloroethene 

 Vinyl chloride 
Table E-1. (continued)

APPENDIX E

E-10

1 Contaminants listed were monitored by at least one Great Lakes state. NOTE: Contaminants monitored exclusively

by the Canadian Province of Ontario were not included. 2 Only the cis-isomer is monitored.

a 301(h) Monitoring Program. Source: U.S. EPA. 1985. Bioaccumulation Monitoring Guidance: 1. Estimating the

Potential for Bioaccumulation of Priority Pollutants and 301(h) Pesticides Discharged into Marine and Estuarine

Waters. EPA 503/3-90-001. Office of Marine and Estuarine Protection, Washington, DC.

b National Study of Chemical Residues in Fish. Source: U.S. EPA. 1992. National Study of Chemical Residues in

Fish. Volumes I and II. EPA 823/R-92-008a and 008b. Office of Science and Technology, Washington, DC.

c Great Lakes Sport Fish Contaminant Advisory Program. Source: Hesse, J. L. 1990. Summary and Analyses of

Existing Sportfish Consumption Advisory Programs in the Great Lakes Basin—the Great Lakes. Fish Consumption

Advisory Task Force, Michigan Department of Health, Lansing, MI.

d NOAA Status and Trends Program. Source: NOAA. 1989. National Status and Trends Program for Marine

Environmental Quality--Progress Report: A Summary of Selected Data on Tissue Contamination from the First Three

Years (1986-1988) of the Mussel Watch Project. NOAA Technical Memorandum NOS OMA 49. U.S. Department of

Commerce, Rockville, MD.

e EPA National Dioxin Study. Source: U.S. EPA. 1987. National Dioxin Study. Tiers 3, 5, 6 and 7. EPA 440/4-87-003.

Office of Water Regulations and Standards, Washington, DC.

f U.S. Fish and Wildlife Service National Contaminant Biomonitoring Program. Sources: C. J. Schmitt, J. L. Zajicek,

and P. H. Peterman. 1990. National Contaminant Biomonitoring Program: Residues of organochlorine chemicals in

U.S. freshwater fish, 1976-1984. Arch. Environ. Contam. Toxicol. 19:748-781; and T. P. Lowe, T. W. May, W. G.

Brumbaugh, and D. A. Kane. 1985. National Contaminant Biomonitoring Program: Concentrations of seven elements

in freshwater fish, 1978-1981. Arch. Environ. Contam. Toxicol. 14:363-388.

g U.S. EPA. 1991. Assessment and Control of Bioconcentratable Contaminants in Surface Waters. Draft. Office of

Water, Office of Research and Development, Washington, DC.

h U.S. Geological Survey National Water-Quality Assessment Program. Source: J.K. Crawford and S.N. Luoma. 1993.

Guidelines for Studies of Contaminants in Biological tissues for the National Water-Quality Assessment Program.

USGS Open-File Report 92-494. U.S. Geological Survey, Lemoyne, PA.
APPENDIX F

PESTICIDES AND HERBICIDES RECOMMENDED

AS TARGET ANALYTES
F-3See notes and references at end of table. 

Table F-1. Pesticides and Herbicides Recommended as Target Analytes

Pesticide Family Use Registration

t

c

Metal Containing PesticidesArsenicals (includingarsenic acid, arsenictrioxide, copperacetoarsenite, leadarsenate, calciumarsenate, sodiumarsenite)

Inorganicarsenicals

A variety of inorganic arsenic compounds are usedas herbicides, fungicides, insecticides androdenticides, but registered uses of some weresuperseded because of their hazard to man andother nontarget species (Farm Chemicals Handbook,

1989)

Some inorganic arsenic compoundregistrations have been canceled; others areunder restricted use application and othersare in special review (U.S. EPA, 1993)

Tributyltins (tribytytinoxide)

Organotins A variety of organotin compounds are used as wood

preservatives, antifoulants, biocides, anddisinfectants (Farm Chemicals Handbook, 1989)

Some organotin compounds have beenactively registered since the mid-1960s. Several registrations have been canceled ormanufacturers discontinued production (U.S.EPA, 1988a)

OrganochlorinesChlordane Chlorinated

cyclodiene

Termite control. Historically used for control of fireants, cutworms, grasshoppers, and on other insectson corn, grapes, strawberries, and other crops and asa dip for nonfood roots and tips of plants (Hartley andKidd, 1987).

In March 1978, EPA issued a cancellationproceeding on chlordane, allowing only limiteduse on certain crops and pests until July1983, but no use thereafter except forunderground termite control (43 FR 12372). All uses were canceled in 1988. 

DDT Chlorinated

hydrocarbon

Insecticide All uses in U.S. were canceled as of

January 1, 1973, except for emergency publichealth uses.

Dicofol Chlorinated

hydrocarbon

Miticide/pesticide on many fruit, vegetable,ornamental, and field crops. Used to control mites oncotton and citrus (80%). Other uses include controlof mites on ornamental plants, fruits, and vegetables. Most of the usage is in California and Florida (U.S.EPA, 1998b). 

Active registration since 1957.c 

Dieldrin Chlorinated

cyclodiene

Formerly used to control locusts, tropical diseasecarriers (e.g., mosquitoes), and termites, use aswood preservative, and moth proofing for woolenclothes and carpets (Worthing, 1991).

All uses on food products were suspended in1974 (ATSDR, 1993). All registered uses inthe U.S. were canceled in 1985.

Endosulfan (I and II) Chlorinated

bicyclid sulfite

Insecticide and acaricide on citrus, deciduous, smallfruits, coffee, tea, fiber crops, forage crops, forest,grains, nuts, oil crops, tobacco, ornamentals, andvegetables (ATSDR, 1999).

Active registration since 1954.c 
F-4

Table F-1. (continued)

Pesticide Family Use Registration

t

c

See notes and references at end of table. 

Endrin Chlorinated

cyclodiene

Historically used to control cotton bollworms, as afoliar treatment for citrus, potatoes, small grains,apple orchards, sugarcane, and as flower and barktreatment on trees. Endrin has also been used tocontrol populations of birds and rodents (U.S. EPA,1980).

In 1964, endrin persistence in soils led tocancellation of its use on tobacco (U.S. EPA,1980). By 1979, specified uses on cotton,small grains, apple orchards, sugarcane andornamentals were also restricted (44 FR43632). All uses in the U.S. were canceled in1984 (U.S. EPA, 1984a).

Heptachlor epoxide Chlorinated

cyclodiene

Heptachlor epoxide is an oxidation product ofheptachlor. It is a contaminant of both heptachlorand chlordane. Heptachlor was widely used as atermiticide and insecticide, primarily for ant control(Hodges, 1977). Chlordane was widely used fortermite control as well as for control of fire ants,cutworms, grasshoppers and other insects (Hartleyand Kidd, 1987).

Restrictions on heptachlor were first institutedin 1978 and heptachlor has not been sold inthe U.S. since August 1987 (ATSDR, 1987).

Hexachlorobenzene Chlorinated

benzene

Primary use prior to 1985 was as a fungicide seedprotectant in small grain crops, particularly wheat.

Registration for all uses was canceled in 1984(Morris and Cabral, 1986).

Lindane( -hexachlorocyclohexane)

Chlorinatedhydrocarbon

Seed treatments, soil treatments for tobaccotransplants, foliage applications on fruit and nut trees,vegetables, and wood and timber protection (ATSDR,1998).

Active registration. Use of lindane in smokefumigation devices for indoor domesticpurposes was banned in 1985 (48 FR 48512,50 FR 5424). Use in dog dips permitted onlyfor veterinary use (U.S. EPA, 1985a). Application permitted only under supervisionof certified applicator (U.S. EPA, 1985a).c

Mirex Chlorinated

cyclodiene

Historically used primarily in fire ant control insoutheastern states (Kutz et al., 1985) and was usedindustrially as a fire retardant and polymerizing agentin plastics under the name dechlorane (ATSDR,1995).

Registration for all uses on field crops wascanceled in 1977 (41 FR 56703) (NAS, 1978).

Toxaphene Chlorinated

camphene

Historically used extensivelyon cotton (Farm Chemicals Handbook, 1989). 

Registration for all uses was canceled in 1982(47 FR 53784) and uses were canceled in1990 (55 FR 31164-31174).
F-5

Table F-1. (continued)

Pesticide Family Use Registration

t

c

See notes and references at end of table. 

OrganophosphatesChlorpyrifos Heterocyclic

organothiophosphate

Insecticide primarily used to control soil and foliarinsect pests on cotton, peanuts, and sorghum(Worthing, 1983; U.S. EPA, 1986a). In addition, it isused to control root-infesting and boring insects on avariety of fruits (e.g., citrus crops, apples, bananas,peaches, grapes, nectarines), nuts (e.g., almonds,walnuts), vegetables (e.g., beans, broccoli, brusselsprouts, cauliflower, soybeans, cabbage, peas) andfield crops (e.g., alfalfa and corn) (U.S. EPA, 1986a)and to control ticks on cattle and sheep (Thomson,1985). As a household insecticide it has been usedto control ants, cockroaches, fleas, and mosquitoes(Worthing, 1983) and is registered for use incontrolling subsurface termites in California (U.S.EPA, 1983).

Active registration since 1965 (U.S. EPA,1984b).c Use patterns will change by the end

of 2001. Virtually all residential use will end,as will use on tomatoes. Use on apples andgrapes will be substantially reduced (U.S.EPA, 2000).

Diazinon Heterocyclic

organothiophosphate

Insecticide and nematicide for control of soil insectsand pests of fruits, vegetables, tobacco, forage, fieldcrops, range, pasture, grasslands, and ornamentals. Used to control cockroaches and other householdinsects; and grubs and nematodes in turf; as a seedtreatment and for fly control (Farm ChemicalsHandbook, 1989).

Active registration since 1952 (U.S. EPA,1986b).c

Disulfoton Aliphatic

organothiophosphate

Systemic insecticide and acaricide on grain, nut,cole, and root crops; pome, strawberry, andpineapple fruits; forage, field and vegetable crops,sugarcane, seed crops, forest plantings, ornamentalsand potted plants (houseplants) (U.S. EPA, 1984c).

Active registration since 1958 (U.S. EPA,1984c).c

Ethion Organothiophosphate Insecticide (nonsystemic) for control of leaf-feeding insects, mites, and scale insects. Citrus accounts for

86%-89% of total pounds of ethion used in the U.S.with the remaining 11%-14% applied to cotton, avariety of fruit trees, nut trees, and vegetables (U.S.EPA, 1989).

Active registration since 1965 (U.S. EPA,1989).c
F-6

Table F-1. (continued)

Pesticide Family Use Registration

t

c

Terbufos Organothiophosphate Systemic insecticide and nematicide on corn, sugar beets, and grain sorghum (U.S. EPA, 1985c). Active since 1974; however, granular end-use products containing 15% or more terbufos

were classified as "Restricted Use" afterSeptember 1985 (U.S. EPA, 1985c; 1985b).c

Chlorophenoxy HerbicidesOxyfluorfen Diphenyl ether Pre- and postemergence herbicide for a wide

spectrum of annual broadleaf weeds and grasses inapples, artichokes, corn, cotton, tree fruit, grapes,nuts, spearmint, peppermint, certain topicalplantation, and ornamental crops (Farm ChemicalsHandbook, 1989)

Active since 1979.c 

a Designations are from EPA (1998a):

I = Oral LD50 up to and including 50 mg/kg in laboratory animals.

II = Oral LD50 >50 through 500 mg/kg in laboratory animals.III = Oral LD50 >500 through 5,000 mg/kg in laboratory animals.IV = Oral LD50 >5,000 mg/kg in laboratory animals.NA = No value available.

b Designations are from IRIS (1999) unless otherwise noted: NA = not available; A = human carcinogen; B1, B2 = probable human carcinogen; C = po

D = not classifiable as to human carcinogenicity; E = evidence of noncarcinogenicity for humans.

c This pesticide has an active registration for agricultural use. The EPA Office of Pesticide Programs is responsible for registration and reregistration of 

Amendment of FIFRA requires EPA to reregister each "registered pesticide containing any active ingredient contained in any pesticide first registered except for any pesticide as to which the Administration has determined, after November 1, 1984 . . . . that—(1) there are no outstanding data requiremrequirements of section 3(c)(5) have been satisfied" (U.S. EPA, 1988b). The Agency will review all relevant data submitted by the registrant for each will use the data to conduct a risk assessment. Any subsequent regulatory action will be based on the results of the risk assessment. If the data subpredetermined review time, the pesticide may be suspended. Under the provisions of the Food Quality Protection Act of 1996, all pesticides with activundergiong review to determine if restrictions on their use are necessary for the protection of the health of infants and children. Contact EPA for furththe review process for specific pesticides.

d EPA carcinogenicity classification based on Reregistration Eligibility Decision (RED) Dicofol (U.S. EPA, 1998b).e EPA carcinogenicity classification based on List of Chemicals Evaluated for Carcinogenic Potential (U.S. EPA, 1999).f EPA carcinogenicity classification based on HEAST, (1997).g EPA carcinogenicity classification based on Revised Human Health Risk Assessment for Chlorpyrifos (U.S. EPA 2000).

Note: The potential human toxicity of currently regulated pesticides is undergoing assessment as a result of provisions of the Food Quality Protection A

the latest assessment information on the Internet at: http://www.epa.gov/oppsrrd1/op/status.htm.
F-7

Table F-1. (continued)

References:ATSDR (Agency for Toxic Substances and Disease Registry). 1987. Draft Toxicological Profile for Heptachlor. U.S. Public Health Service, WashingtonATSDR (Agency for Toxic Substances and Disease Registry). 1993. Toxicological Profile for Aldrin/Dieldrin (Update). U.S. Public Health Service, WasATSDR (Agency for Toxic Substances and Disease Registry). 1995. Toxicological Profile for Mirex/Chlordecone.. U.S. Public Health Service, Washing

ATSDR (Agency for Toxic Substances and Disease Registry). 1998. Toxicological Profile for Alpha-, Beta-, Gamma-, and Delta-Hexachlorocyclohexane 

Service, Washington, DC.

ATSDR (Agency for Toxic Substances and Disease Registry). 1999. Draft Toxicological Profile for Endosulfan. U.S. Public Health Service, Washington

Farm Chemicals Handbook. 1989. Meister Publishing Company, Willoughby, OH.Hartley, D., and H. Kidd (eds.). 1987. Agrochemicals Handbook. Royal Society of Chemistry, Nottingham, England.

Hodges, L. 1977. Environmental Pollution. Holt, Rinehart and Winston, New York, NY.IRIS (Integrated Risk Information System). 1999. U.S. Environmental Protection Agency, Duluth, MN.Kutz,F.W., S.C. Strassman, C.R. Stroup, J.C. Carra, C.C. Leininger, D.L. Watts, and C.M. Sparacino. 1985. The human body burden of mirex in the so

Toxicol. and Environ. Health 15:385-394.

Morris, C.R., and J.R.P. Cabral (eds.). 1986. Hexachlorobenzene: Proceedings of an International Symposium. IARC Scientific Publication No. 77. W

Lyon, France.

NAS (National Academy of Sciences). 1978. Kepone/Mirex/Hexachlorocyclopentadiene: An Environmental Assessment. National Academy of Science

Council, Washington, DC.

Thomson, W.T. 1985. Agricultural Chemicals Book I - Insecticide, 1985 revision. Thomas Publication, Davis, CA.U.S. EPA (U.S. Environmental Protection Agency). 1980. Ambient Water Quality Criteria for Endrin. EPA-440/5-80-047. Office of Water Regulations a

DC.

U.S. EPA (U.S. Environmental Protection Agency). 1983. Analyses of the Risks and Benefits of Seven Chemicals Used for Subterranean Termite Cont

Office of Pesticide Programs, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1984a. Internal memorandum from G. LaRocca to B. Burnam et al., August 16, 1984. Office of Pes

DC.

U.S. EPA (U.S. Environmental Protection Agency). 1984b. Pesticide Fact Sheet—Chlorpyrifos. Office of Pesticides and Toxic Substances, Office of Pe

Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1984c. Pesticide Fact Sheet—Disulfoton. Office of Pesticides and Toxic Substances, Office of Pes

Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1985a. Guidance for the Registration of Pesticide Products Containing Lindane as the Active Ingred

U.S. EPA Office of Pesticide Programs. Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1985b. Suspended, Cancelled, and Restricted Pesticides. U.S. EPA Office of Pesticides and Toxic 

DC.

U.S. EPA (U.S. Environmental Protection Agency). 1985c. Pesticide Fact Sheet—Terbufos. Office of Pesticides and Toxic Substances, Office of Pesti

Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1986a. Ambient Water Quality Criteria for Chlorpyrifos. EPA-440/5-86-005. Office of Water Regula

Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1986b. Pesticide Fact Sheet—Diazinon. Office of Pesticides and Toxic Substances, Office of Pesti

Washington, DC.
F-8

Table F-1. (continued)

U.S. EPA (U.S. Environmental Protection Agency). 1988a. Pesticide Fact Sheet—Tributyltin Antifouling Paint. Office of Pesticides and Toxic Substanc

Programs, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1988b. The Federal Insecticide, Fungicide, and Rodenticide Act as Amended. EPA-540/09-89-012. 

Programs, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1989. Pesticide Fact Sheet—Ethion. Office of Pesticides and Toxic Substances, Office of Pesticide 

U.S. EPA (U.S. Environmental Protection Agency). 1990. Suspended, Cancelled, and Restricted Pesticides. Document 20T-1002, Office of Pesticides 

Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1993. Status of Pesticides in Reregistration and Special Review. Prevention, Pesticides and Toxic 

009. Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1998a. Health Effects Test Guidelines, OPPTS 870.1000. Acute Toxicity Testing—Background: Pr

Toxic Substances. EPA-712-C-98-189. Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1998b. Reregistration Eligibility Decision for Dicofol. Office of Pesticide Programs, Washington, DC

U.S. EPA (U.S. Environmental Protection Agency). 1999. List of Chemicals Evaluated for Carcinogenic Potential. Office of Pesticide Programs, Health 

DC.

U.S. EPA (U.S. Environmental Protection Agency). 2000. Revised Human Health Risk Assessment for Chlorpyrifos. Office of Pesticide Programs, Was

Worthing, C.R. 1991. The Pesticide Manual: A World Compendium. 9th edition. British Crop Protection Council, Croydon, England.
APPENDIX G

TARGET ANALYTE DOSE-RESPONSE

VARIABLES AND ASSOCIATED INFORMATION
G-3See notes and references at end of table. 

Table G-1. Target Analyte Dose-Response Variables and Associated Information

Target analyte

Noncarcinogens Carcinogens

RfDa

(degree of

confidence;

uncertainty

factor) Critical toxic effect

CSFb

(discussion of

confidence)

Critical

carcinogenic

effectc

Metals

Arsenic (inorganic) 3 × 10!4

(medium; 3)

Hyperpigmentation,keratosis and possiblevascular complications inhumans

1.5 — 

Cadmium 1 × 10!3

(high; 10)

Significant proteinurea inhumans

NA — 

Mercury (asmethylmercury)

1 × 10!4 e(medium; 10)

Developmental neurological abnormalities inhuman infants

NA — 

Seleniumf 5 × 10!3

(high; 3)

Selenosis in humans NA — 

Tributylting 3 x 10-4

(high; 100)

Immunotoxicity in rats NA — 

Organochlorine Pesticides

Chlordane (sum of cisand trans-chlordane, cisand trans-nonachlor, andoxychlordane)h

5 × 10!4

(medium; 300)

Hepatic necrosis in mice 0.35

(Adequate number of

animals observed. CSF

is the geometric mean

of CSFs for five data

sets).

Hepatocellular carcinom

5 strains of mice (male 

female)

DDT (sum of 4,4'- and2,4'- isomers of DDT,DDE, and DDD)i

5 × 10!4

(medium; 100)

Liver lesions in rats 0.34

(CSF is geometric

mean of CSFs from 10

data sets.

DDT: Liver tumors in se

studies in various mou

strains and three studie

three rat strains
G-4

Table G-1. (continued)

Target analyte

Noncarcinogens Carcinogens

RfDa

(degree of

confidence;

uncertainty

factor) Critical toxic effect

CSFb

(discussion of

confidence)

Critical

carcinogenic

effectc

See notes and references at end of table. 

Dicofol 4 x 10-4 j

(NA, 300)

Inhibition of ACTHstimulated release ofcortisol in both sexes in1-yr dog feeding study.

NA — 

Dieldrin 5 × 10-5

(medium; 100)

Liver lesions (focalproliferation and focalhyperplasia) in one strainof female rats

16

(CSF is the geometric

mean of CSFs from 13

data sets. Individual

CSFs ranged within a

factor of 8.)

Liver carcinomas in fiv

strains of mice (male a

female)

Endosulfan (sum of endosulfan I andII)

6 × 10-3

(medium; 100)

Decreased body weightgain in male and femalerat and progressiveglomerulonephrosis andblood vessel aneurysms inone strain of male rats

NA — 

Endrin 3 × 10-4

(medium; 100)

Mild histological lesions inlivers, occasional convulsions in dogs (both sexes)

NA — 

Heptachlor epoxide 1.3 × 10-5

(low; 1000)

Increased liver-to-bodyweight ratios in male andfemale dogs

9.1

(Adequate number of

animals observed in

both studies, but

survival in one study

was low. This CSF is

consistent with CSF =

5.8 for one strain of

seven rats.)

Hepatocellular carcinom

two strains of mice (male 

female)
G-5

Table G-1. (continued)

Target analyte

Noncarcinogens Carcinogens

RfDa

(degree of

confidence;

uncertainty

factor) Critical toxic effect

CSFb

(discussion of

confidence)

Critical

carcinogenic

effectc

See notes and references at end of table. 

Hexachlorobenzene 8 × 10-4

(medium; 100)

Liver effects (hepaticcentrilobular basophilicchromogenesis) in onestrain of rats (both sexes)

1.6

(Significant increases in

malignant tumors

observed among an

adequate number of

animals observed for

their lifetime.)

Hepatocellular carcinom

one strain of rats (fema

only)

Lindane (-HCH) 3 × 10-4

(medium; 1,000)

Liver and kidney toxicity(liver hypertrophy, kidneytubular degeneration,hyaline droplets, tubulardistension, interstitialnephritis, and basophilictubules) in both sexes ofone strain of rats

1.3m — 

Mirex 2 × 10-4

(high; 300)

Liver cytomegaly, fattymetamorphosis,angiectasis and thyroidcystic follicles in one strainof rats.

NA — 

Toxaphene 2.5 x 10-4 n

(NA, 1,000)

Slight liverdegeneration—granularityand vacuolization ofhepatocytes.

1.1

(Adequate number of

animals observed. A

dose-response effect

was seen in a study

with three non-zero

dose levels.)

Hepatocellular carcinom

and neoplastic nodules 

one strain of mice (ma

only)
G-6

Table G-1. (continued)

Target analyte

Noncarcinogens Carcinogens

RfDa

(degree of

confidence;

uncertainty

factor) Critical toxic effect

CSFb

(discussion of

confidence)

Critical

carcinogenic

effectc

See notes and references at end of table. 

OrganophosphatePesticides

Chlorpyrifos 3 x 10-4 o

(NA, 10)

Decreased plasma ChEactivity observed in variousanimal feeding studies.

NA — 

Diazinon 7 x 10-4 p

(NA, 30)

Inhibition of plasma ChEobserved in 90-d ratfeeding study.

NA — 

Disulfoton 4 x 10-5

(medium, 100)

ChE inhibition anddegeneration of the opticnerve observed in 2-yr ratfeeding study.

NA — 

Ethion 5 x 10-4

(medium, 100)

Plasma ChE inhibition (in21-d human feeding study)and inhibition of brain ChEobserved in 90-d dogfeeding study.

NA — 

Terbufos 2 x 10-5 q

(NA, 300)

Inhibition of plasma ChEobserved in 28-d dogfeeding study.

NA — 

Chlorophenoxy Herbicides

Oxyfluorfen 3 x 10-3

(high, 100)

Increased absolute liverweight and nonneoplasticlesions observed in 20-momouse feeding study.

7.32 x 10-2 l Evidence of carcinogen

(liver tumors) in mice
G-7

Table G-1. (continued)

Target analyte

Noncarcinogens Carcinogens

RfDa

(degree of

confidence;

uncertainty

factor) Critical toxic effect

CSFb

(discussion of

confidence)

Critical

carcinogenic

effectc

See notes and references at end of table. 

PAHsr

Benzo[a]pyrene NA — 7.3

(Data less than optimal,

but acceptable. Four

data sets used from two

different studies using

two different species

(rats and mice; both

sexes) to derive

geometric mean of four

calculated slope

factors.)

Squamous cell carcinom

the forestomach in one s

of mice (both sexes)

Forestomach, larynx, a

esophagus papillomas 

carcinomas in one strai

rats (both sexes)

PCBs

Total PCBs (sum ofAroclors)

2 x 10-5 s

(medium; 300)

Ocular exudate, inflamed,prominent Meibomianglands, distorted growth offingernails, and toenails,decreased antibodyresponse to sheeperythrocytes in monkeyclinical and immunologicstudies

2.0t

(Adequate number of

animals observed for

their normal lifespan. 

Only one non-zero test

dose used.)

Trabecular

carcinomas/adenocarci

mas, neoplastic nodule

one strain of rats (fema

only)

Dioxins/furans NA — 1.56 × 105 u NA 

NA = Not available in IRIS (1992).PAHs = Polycyclic aromatic hydrocarbons.PCBs = Polychlorinated biphenyls.

DDT = p,p’-Dichlorodiphenyl trichloroethane.

DDD = p,p’-Dichlorodiphenyl dichloroethylene.

DDE = p,p’-Dichlorodiphenyl trichloroethane.
G-8

Table G-1. (continued)

a RfD = Oral reference dose (mg/kg-d); from IRIS (1999) unless otherwise noted (see Section 5.1.1).b CSF = Oral cancer slope factor (mg/kg-d)!1; from IRIS (1999) unless otherwise noted (see Section 5.1.2).c The critical effect is the effect observed in oral dose response studies used to determine the CSF.d Except where noted, all EPA carcinogenicity classifications are taken from IRIS (1999):

A = Human carcinogen based on sufficient evidence from epidemiologic studies.B1 = Probable human carcinogen based on limited evidence of carcinogenicity to humans.B2 = Probable human carcinogen based on sufficient evidence in animals and inadequate or no data in humans.C = Possible human carcinogen based on limited evidence of carcinogenicity in animals in the absence of human data.D = Not classifiable based on lack of data or inadequate evidence of carcinogenicity from human or animal data.E = No evidence of carcinogenicity for humans (no evidence of carcinogenicity in at least two adequate animal tests in different 

epidemiologic and animal studies).

e The RfD for methylmercury should be considered an interim value. The National Academy of Sciences (NAS) conducted an indepen

RfD and concluded, “On the basis of its evaluation, the committee consensus is that the value of EPA’s current RfD for a scientificallprotection of human health.” (NAS 2000).

f The evidence of carcinogenicity for various selenium compounds in animals and mutagenicity studies is conflicting and difficult to int

evidence for selenium sulfides is sufficient for a B2 classification (IRIS, 1999).

g The oral RfD and cancer classification are for tributyltin oxide (IRIS, 1999).h The RfD and CSF values listed are derived from studies using technical-grade chlordane (IRIS, 1999) for the cis- and trans-chlordan

chlordane metabolite, oxychlordane, or for the chlordane impurities cis- and trans-nonachlor. It is recommended that the total chlorda

determined by summing the individual concentrations of cis- and trans-chlordane, cis- and trans-nonachlor, and oxychlordane.

i The RfD value listed is for DDT. The CSF value is for total DDT (sum of DDT, DDE, and DDD) or DDE; the CSF value for DDD is 0.2

Carcinogenicity Assessment Group recommended the use of CSF = 0.34 for any combination of DDT, DDE, DDD, and dicofol (Holderecommended that the total concentration of the 2,4'- and 4,4'-isomers of DDT and its metabolites, DDE and DDD, be determined. 

j The RfD value is from a memorandum dated December 12, 1997. Dicofol: Report of the Hazard Identification Assessment Review C

Document No. 012439 (U.S. EPA, 1997b).

k EPA carcinogenicity classification based on Reregistration Eligibility Decision (RED) Dicofol (U.S. EPA, 1998b).l EPA carcinogenicity classification based on U.S. EPA, (1999).m EPA CSF based on HEAST (1997).n Reference dose information is taken from the Office of Pesticide Programs Reference Dose Tracking Report (U.S. EPA, 1997a).o Oral RfD based on the Revised Human Health Risk Assessment for Chlorpyrifos (U.S. EPA, 2000).p The RfD value is from a memorandum dated April 1, 1998, Diazinon: Report of the Hazard Identification Assessment Review Comm

012558 (U.S. EPA, 1998a).

q The RfD value listed is from a memorandum dated September 25, 1997; Terbufos-FQPA Requirement- Report of the Hazard Identifi

1997c).
G-9

Table G-1. (continued)

r This CSF is for benzo[a]pyrene (IRIS, 1999). There are no other RfDs or CSFs listed for other PAHs in IRIS (1999). It is recommend

be analyzed for benzo[a]pyrene and 14 other PAHs (Nisbet and LaGoy, 1992; U.S. EPA, 1993) and that the order-of-magnitude relat

these PAHs be used to calculate a potency equivalency concentration (PEC) for each sample for comparison with the recommended 

(see Section 5.3.2.4). 

s This RfD for PCBs is based on the chronic toxicity of Aroclor 1254 (IRIS, 1999).t This CSF is based on a carcinogenicity assessment of Aroclor 1260, 1254, 1242, and 1016. The CSF represented is the upper boun

chain exposure. The central estimate is 1.0 (IRIS, 1999). 

u The CSF value listed is for 2,3,7,8-tetrachlorodibenzo-p-dioxin 2,3,7,8-TCDD (HEAST, 1997). It is recommended that, in both screen

the 17 tetra- through octa-chlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and the 12 dioxin-like PCBs be determweighted total concentration be calculated for each sample for comparison with the recommended SV, using the method for estimatiConcentration (TEQ) (Van den Berg et al., 1998). 

References:HEAST. 1997. Health Effects Summary Tables. Office of Emergency and Remedial Response, U.S. Environmental Protection Agency

Holder, J.W. 1986. The Assessment of the Carcinogenicity of Dicofol (Kelthane), DDT, DDE, and DDD (TDE). EPA-600/6-86/001. Car

Group, Office of Pesticide Programs, U.S. Environmental Protection Agency, Washington, DC. 

IRIS (Integrated Risk Information System). 1999. U.S. Environmental Protection Agency, Duluth, MN. NAS (National Academy of Sciences). 2000. Toxicological Effects of Methylmercury. National Research Council, Washington, DC.

Nisbet and LaGoy. 1992. Toxic equivalency factors (TEFs) for polycyclic aromatic hydrocarbons (PAHs). Reg. Toxicol. Pharmacol. 16U.S. EPA (U.S. Environmental Protection Agency). 1993. Provisional Guidance for Quantitative Risk Assessment of Polycyclic Aromat

EPA/600/R-93/089. Environmental Criteria and Assessment Office, Office of Health and Environmental Assessment, Cincinnati, OH

U.S. EPA (U.S. Environmental Protection Agency). 1997a. Reference Dose Tracking Report. Office of Pesticide Programs, Health Eff

DC.

U.S. EPA (U.S. Environmental Protection Agency). 1997b. Memorandum dated December 12, 1997. Dicofol: Report of the Hazard Id

Review Committee.. HED DOC No. 012439. Office of Pesticide Programs, Health Effects Division, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1997c. Terbufos-FQPA Requirement–Report of the Hazardous Assessment Identifi

Pesticide Programs, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1998a. Memorandum dated April 1, 1998. Diazinon: Report of the Hazard Identific

Committee. HED DOC No. 012558. Office of Pesticide Programs, Health Effects Division, Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1998b. Reregistration Eligibility Decision for Dichofol. Office of Pesticide Programs 

Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 1999. Classification List of Chemical Evaluated for Carcinogenicity Potential. Offic

Washington, DC.

U.S. EPA (U.S. Environmental Protection Agency). 2000. Revised Human Health Risk Assessment for Chlorpyrifos. Office of Pesticid

DC.

Van den Berg, et al. 1998. Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for human and wildlife. Environ. Health Perspe
APPENDIX H

A RECOMMENDED METHOD FOR

INORGANIC ARSENIC ANALYSIS

Extracted from:

Crecelius, E.A., N.S. Bloom, C.E. Cowan, and E.A. Jenne. 1986. Speciation

of Selenium and Arsenic in Natural Waters and Sediments. Volume 2: 

Arsenic Speciation, Section 2, in EPRI report #EA-4641, Vol. 2, pp. 2–1

to 2–28.
APPENDIX H

H-3

APPENDIX H

A RECOMMENDED METHOD FOR

INORGANIC ARSENIC ANALYSIS

Note: EPA is currently revising Method 1632: Determination inorganic arsenic

in water by hydride generation flame atomic absorption to include fish

tissue.
2-1

Section 2

DETERMINATION OF ARSENIC SPECIES IN LIMNOLOGICAL SAMPLES

BY HYDRIDE GENERATION ATOMIC ABSORPTION SPECTROSCOPY

INTRODUCTION

This section describes the analytical methods used to determine the arsenic species in waters and

sediments. Also, sample storage tests were conducted to select methods of storing and shipping

environmental samples that would minimize changes in speciation. Based on results of previous

studies we selected hydride generation coupled with atomic absorption spectroscopy as ithe

method of quantification of arsenic. In this technique arsenate, arsenite, methylarsonic acid, and

dimethylarsinic acid are volatilized from solution at a specific pH after reduction to the

corresponding arsines with sodium borohydride (1). The volatilized arsines are then swept onto a

liquid nitrogen cooled chromatographic trap, which upon warming, allows for a separation of

species based on boiling points. The released arsines are swept by helium carrier gas into a quartz

cuvette burner cell (2), where they are decomposed to atomic arsenic. Arsenic concentrations are

determined by atomic absorption spectroscopy. Strictly speaking, this technique does not

determine the species of inorganic arsenic but rather the valence states of arsenate (V) and

arsenite (III). The actual species of inorganic arsenic are assumed to be those predicted by the

geochemical equilibrium model described in Section 1 of this report. 

EXPERIMENTAL SECTION

Apparatus

The apparatus needed for the volatilization, separation and quantitation of arsenic species is shown

schematically in Figure 2-1-a. Briefly, it consists of a reaction vessel, in which arsenic compounds

are reduced to volatile arsines, a liquid nitrogen cooled gas chromatographic trap, and a H-2 flame

atomic absorption detector. 

Reaction Vessel. The reaction vessel is made by grafting a side-arm inlet onto a 30-ml "Midget

Impinger" (Ace Glass #7532-20), as illustrated in Figure 2-1-b. The 8-mm diameter side arm may

then be sealed with a silicone rubber-stopper type septum (Ace Glass #9096-32) to allow the airfree injection of sodium borohydride. The standard impinger assembly is replaced with a 4-way

Teflon stopcock impinger (Laboratory Data control #700542) to allow rapid and convenient

switching of the helium from the purge to the analysis mode of operation. 

GC Trap. The low temperature GC trap is constructed from a 6 mm o.d. borosilicate glass U-tube

about 30-cm long with a 2-cm radius of bend (or similar dimensions to fit into a tall widemouth

Dewar flask. Before packing the trap, it is silanized to reduce the number of active adsorption sites

on the glass. This is accomplished using a standard glass silanizing compound such as Sylon-Ct®

(Supelco Inc.). The column is half-packed with 15% 0V-3 on Chromasorb® WAW-DMCS (45-60
2-2

mesh). A finer mesh size should not be used, as the restriction of the gas flow is sufficient to

overpressurize the system. After packing, the ends of the trap are plugged with silanized glass

wool. 

The entire trap assembly is then preconditioned as follows: The input side of the trap (nonpacked

side) is connected via silicone rubber tubing to helium at a flow rate of 40-ml • min-1 and the whole

assembly is placed into an oven at 175°C for 2 hours. After this time, two 25-μl aliquots of GC

column conditioner (Silyl-8®, Supelco Inc.) are injected by syringe through the silicone tubing into

the glass tubing. The column is then left in the oven with helium flowir,g through it for 24 hours.

This process, which further neutralizes active adsorption sites and purges the system of foreign

volatiles, may be repeated whenever anaiate peaks are observed to show broadening. 

Once the column is conditioned, it is evenly wrapped with about 1.8 m of nichrome wire (22 gauge)

the ends of which are affixed to crimp on electrical contacts. The wire-wrapped column is then

coated about 2-mm thick all over with silicone rubber caulking compound and allowed to dry

overnight. The silicone rubber provides an insulating layer which enhances peak separation by

providing a longer temperature ramp time. 

The wnpacked side of the column is connected via silicone rubber tubing to the output from the

reaction vessel. The output side of the trap is connected by a nichrome-wire wrapped piece of 6-

mm diameter borosilicate tubing to the input of the flame atomizer. It is very important that the

system be heated everywhere (~80°C) from the trap to the atomizer to avoid the condensation of

water. Such condensation can interfere with the determination of dimethylarsine. All glass-to-glass

connections in the system are made with silicone rubber sleeves. 

Atomizer. The eluted arsines are detected by flame atomic absorption, using a special atomizer

designed by Andreae (2). This consists of a quartz cross tube as shown in Figure 2-1-c. Air is

admitted into one of the 6-mm o.d. side tubes (optimal flows are given in Table 2-1), while a mixture

of hydrogen and the carrier gas from the trap is admitted into the other. This configuration is

superior to that in which the carrier gas is mixed with the air (Andreae, personal communication

1983) due to the reduction of flame noise and possible extinguishing of the flame by

microexplosions when H2 is generated in the reaction vessel. To light the flame, all of the gases

are turned on, and a flame brought to the ends of the quartz cuvette. At this point a flame will be

burning out of the ends of the tube. After allowing the quartz tube to heat up (~5 minutes) a flat

metal spatula is put smoothly first over one end of the tube, and then the other. An invisible

air/hydrogen flame should now be burning in the center of the cuvette. This may be checked by

placing a mirror near the tube ends and checking for water condensation. Note that the flame must

be burning only inside the cuvette for precise, noise-free operation of the detector. 

Precision and sensitivity are affected by the gas flow rates and these must be individually optimized

for each system, using the figures in Table 2-1 as an initial guide. We have observed that as the

O2/H2 ratio goes up, the sensitivity increases and the precision decreases. As this system is

inherently very sensitive, adjustments are made to maximize precision. 

Detector. Any atomic absorption unit may serve as a detector, once a bracket has been built to hold

the quartz cuvette burner in the wave path. This work has been done using a Perkin-Elmer Model

5000® spectrophotometer with electrodeless discharge arsenic lamp. An analytical wavelength of

197.3 nm and slit width of 0.7 nm (low) are used throughout. This wavelength has been shown to

have a longer linear range, though about half the sensitivity of the 193.7 nm line (2). Background
2-3

correction is not used as it increases the system noise and has never been found necessary on the

types of sample discussed in this paper. 

Standards and Reagents

Arsenite (As(III)) Standards. A 1000 • mg l-1 stock solution is made up by the dissolution of 1.73

grams of reagent grade NaAsO2 in 1.0-liter deionized water containing 0.1% ascorbic acid. This

solution is kept refrigerated in an amber bottle. A 1.0 mg • l-1 working stock solution is made by

dilution with 0.1% ascorbic acid solution and stored as above. Under these conditions this solution

has been found stable for at least one year. 

Further dilutions of As(III) for analysis, or of samples to be analyzed for As(III), are made in filtered

Dungeness River water. It has been observed both here and elsewhere (Andreae 1983) that

deionized water can have an oxidizing potential that causes a diminished As(III) response at low

levels (1 μg l-1 and less). Dilute As(III) standards are prepared daily. 

Arsenate (As(V)) Standards. To prepare a 1000 mg • l-1 stock solution, 4.16 g of reagent grade

Na2HASO4 • 7H2O are dissolved in 1.0 liter of deionized water. Working standards are prepared

by serial dilution with deionized water and prepared monthly. 

Monomethylarsonate (MMA) Standards. To prepare a stock solution of 1000 mg • l-l, 3.90 g of

CH3AsO(ONa)2 • 6H2O is dissolved in 1.0 liter of deionized water. Working standards are prepared

by serial dilution with deionized water. Dilute standards are prepared weekly. 

Dimethylarsinate (DMA) Standards. To prepare a stock solution of 1000mg 1-l, 2.86 g of reagent

grade (CW3)2AsO2Na 3H2O (cacodylic acid, sodium salt) is dissolved in 1.0 liter deionized water.

Dilute standards are handled as for MMA. 

6M Hydrochloric Acid. Equal volumes of reagent grade concentrated HCl and deionized water are

combined to give a solution approximately 6M in HCl. 

Tris Buffer. 394 g of Tris HCl (tris (hydroxymethyl) aminomethane hydrochloride) and 2.5 g of

reagent grade NaOH are dissolved in deionized water to make 1.0 liter. This solution is 2.5 M in

tris and 2.475 M in HCl, giving a pH of about 6.2 when diluted 50-fold with deionized water. 

Sodium Borohydride Solution. Four grams of >98% NaBH4 (previously analyzed and found to be

low in arsenic) are dissolved in 100 ml of 0.02 M NaOH solution. This solution is stable 8-10 hours

when kept covered at room temperature. It is prepared daily. 

Phosphoric Acid Leaching Solution. To prepare 1.0 liter of 0.10 M phosphoric acid solution, 6.8 ml

of reagent grade 85% H3PO4 are dissolved in deionized water. 

Trisodium Phosphate Leaching Solution. To prepare 1.0 liter of 0.10 M trisodium phosphate

solution, 6.8 ml of 85% H3PO4 and 12 g of reagent grade NaOH are dissolved in deionized water.

Acid Digestion Mixture. With constant stirring, 200 ml of concentrated reagent grade H2SO4 are

slowly added to 800 ml concentrated HNO3. 
2-4

METHODS

Total Arsenic Determination

An aqueous sample (5-30 ml) is placed into the reaction vessel and 1.0 ml of 6M HCl is added. The

4-way valve is put in place and turned to begin purging the vessel. The G.C. trap is lowered into

a Dewar flask containing liquid nitrogen (LN2) and the flask topped off with LN2 to a constant level.

A 2.0-ml aliquot of NaBH4 solution is then introduced through the silicone rubber septum with a

disposable 3-ml hypodermic syringe and the timer turned on. The NaBH4 is slowly added over a

period of about 1 minute, being careful that the H2 liberated by the reduction of water does not

overpressurize the system or foam the contents out of the reaction vessel. 

After purging the vessel for 8 minutes, the stopcock is turned to pass helium directly to the G.C.

trap. In rapid order, the LN2 flask is removed, the trap heating coil is turned on, and the chart

recorder is turned on. The arsines are eluted in the order: AsH3, CH3AsH2, (CH3)2AsH according

to their increasing boiling points given in Table 2.2 (1). 

Arsenic (III) Determination

The same procedure as above is used to determine arsenite, except that the initial pH is buffered

at about 5 to 7 rather than <1, so as to isolate the arsenous acid by its pKa (1). This is

accomplished by the addition of 1.0 ml of Tris buffer to a 5- to 30-ml aliquot of unacidified sample.

(If the sample is acidic or basic, it must be neutralized first, or the buffer will be exhausted.) For the

As(III) procedure, 1.0 ml of NaBH4 is added in a single short (~10 seconds) injection, as the rapid

evolution of H2 does not occur at this pH. 

Small, irreproducible quantities of organic arsines may be released at this pH and should be

ignored. The separation of arsenite, however, is quite reproducible and essentially 100% complete.

As(V) is calculated by subtracting the As(III) determined in this step from the total inorganic arsenic

determined on an aliquot of the same sample previously. 

SEDIMENTS

Total Inorganic Arsenic

A 1.00-g aliquot of freeze-dried and homogenized sediment is placed into a 100-ml snap-cap

volumetric flask. Five milliliters of deionized water is added to form a slurry and then 7 ml of the

acid digestion mixture is added. After 5 minutes, the caps are replaced and the flasks heated at

80 to 90°C for 2 hours. Upon cooling the samples are diluted to the mark with deionized water,

shaken, and allowed to settle overnight. An appropriate-sized aliquot of the supernatant liquid (25-

100 μl) is added to 20 ml of deionized water and run as for total arsenic. 

Leachable Arsenite

An aliquot (~1-2 g) of fresh or freshly thawed wet homogeneous sediment is weighed to the nearest

10 mg directly into a 40-ml acid-cleaned Oak Ridge type centrifuge tube. To this is added 25 ml of

0.10 M H3PO4 solution and the tubes are agitated with the lids on. Periodic agitation is maintained
2-5

for 18 to 24 hours, at which time the tubes are centrifuged for 30 minutes at 2500 RPM. Twenty

milliliter aliquots of the supernatant liquid are removed by pipetting into cleaned polyethylene vials

and saved in the refrigerator until analysis. Analysis should be accomplished within the next couple

days. 

For analysis, an appropriate-sized aliquot (10-100 μ1) is added to 20 ml of well-characterized

filtered river water (or other nonoxidizing/nonreducing water). Enough 1.0 M NaOH solution is

added to approximately naturalize the H3PO4 (1/3 the volume of the sample aliquot), and then 1.0

ml of Tris buffer is added. The sample is then analyzed as for As(III). 

Leachable Arsenate, MMA and DMA

An aliquot (~1-2 g) of wet sediment is weighed into a centrifuge tube, as above. To this are added

25 ml of 0.1 M Na3PO4 solution, and the tubes agitated periodically for 18 to 24 hours. After

centrifugation the supernatant liquid (dark brown due to released humic materials) is analyzed as

for total arsenic using an appropriate-sized aliquot in 20 ml of deionized water. The total inorganic

arsenic in this case should be only As(V), as As(III) is observed to not be released at this pH. No

pre-neutralization of the sample is necessary as the HCl added is well in excess of the sample

alkalinity. 

Interstitial Water Analysis

Interstitial water samples may be treated just as ordinary water, except that as they are quite high

in arsenic, usually an aliquot of 100 to 1000 μl diluted in deionized water or river water is

appropriate in most cases. 

Storage Experiments

Storage experiments designed to preserve the original arsenic speciation of samples were carried

out for a wide variety of conditions. For water samples, 30-ml and 60-ml polyethylene bottles

precleaned in 1 M HCl were used. 

Conditions of temperature ranging from 20°C to -196°C were assessed, as well as preservation

with HCl and ascorbic acid. Storage tests were carried out over a period of one month for water

samples. 

The stability of the As(III)/As(V) ratio in interstitial water at room temperature, in the presence ot

air was carried out over a 24-hour period to determine the feasibility of the field collection of

interstitial water. 

Because of the time-consuming nature of sediment analysis, a two-point storage test was carried

out with triplicate samples analyzed for two sediments at two temperatures (0°C and -18°C). Mud

samples were stored in polyethylene vials and analyzed at time zero and one month. 
2-6

RESULTS AND DISCUSSION

Data Output

Using the procedures outlined above, and a mixed standard containing As(V), MMA, and DMA,

standard curves were prepared for each of the arsines generated. A typical chromatogram from

this procedure is illustrated in Figure 2.2. Under the cor,ditions described in this paper, the elusion

times for the various arsines are as follows: AsH3, 24 ± 2 s; CH3AsH2, 53 ± 2 s; and (CH3)2AsH, 66

± 2 s. Notice that the peaks are broadened and that the sensitivity decreases as the boiling point

of the compound increases. The small amount of signal after the DMA peak is probably a higher

boiling impurity in the DMA, or some DMA that is lagging in the system during elusion. We had

previously noted much larger, multiple peaks in this region when water was allowed to condense

between the trap and the detector. Such peaks were effectively eliminated and the DMA peak

sharpened with the addition of the heating coil between the trap and the detector. 

The typical standard curves in Figure 2.3 are prepared from the mean of two determinations at

each concentration. Arsenic peak-height response appears to be linear to at least 600 mau

(milliabsorbance units), which is the full scale setting used on our chart recorder. Andreae (3)

shows that arsenic response is extremely nonlinear above this for the peak height mode, and

recommends the use of peak area integration to increase the linear range. We have chosen to

simply use a small enough sample aliquot to remain within 600 mau. 

As arsenic response is quite sensitive to the H2/O2 ratio in the flame, it is necessary to

restandardize the instrument whenever it is set up. Usually, however, the response is quite

constant and stable over the entire day. 

Precision, Accuracy, and Detection Limits

Precision and accuracy are the greatest and the detection limits the lowest for inorganic arsenic.

The precision and accuracy of the inorganic arsenic determination is illustrated at two

concentrations in Table 2-3. The standard seawater, NASS-1 (National Research Council of

Canada) was run in 5.0-ml aliquots and the "standard river water" (National Bureau of Standards)

was run in 100-μl aliquots. In either case, both the precision (RSD) and accuracy were about 5%.

Precision begins to decrease, as the boiling point of the compound increases, as is illustrated in

Table 2-4, for spiked river water. No standard reference material has been found for the organic

species. 

The detection limit of this technique has not been explored to the extreme as the usual

environmental sample benefits from less, not more sensitivity. For a chart recorder expansion of

600 mau full scale, and the parameters given in the text, and for a 30-ml sample aliquot, the

following approximate detection limits are found: As(V), 0.006 μg • 1-1 (twice the standard deviation

of the blank); As(III) 0.003 μg • 1-1 (0.5 chart units); MMA, 0.010 μg • 1-1 as As (0.5 chart units);

DMA, 0.012 μg • 1-1 as As (0.5 chart units). For As(III), MMA and DMA, no contribution to the blank

has been found due to reagents, except for the As(III) present in the river water used as a dilutant.

As for As(V) a small contribution is found, mostly from the NaBH4, and to a smaller extent from

H3PO4. These may be minimized by selecting reagent lots of reagents found to be low in arsenic.
2-7

Water Storage Experiments

From the many experiments undertaken to determine a storage regime for arsenic species, the

following general conclusion can be made: Almost any storage scheme will preserve the total

arsenic, MMA, and DMh concentrations of river water in the μ • 1-1 range. This is illustrated in the

Figures 2-4a-p, where the final concentration of these parameters was within ±20% of the initial

in all cases. The noise in the data is due mostly to the day-to-day analytical variability, which has

been observed to be about twice that of same-day replicate analysis. On the other hand, these data

also show that it is very difficult to preserve the original As(III)/As(V) ratio in samples, even for a

short time. Two major observations are made: first, river water (0ungeness River water) tends to

spontaneously reduce As(V) to As(III), even though the water has been filtered to 0.4 ~, thus

removing most living creatures. This is also curious, as the natural equilibrium As(III)/As(V) ratio

is about 0.2 in Dungeness River water. It is surmised that dissolved organic materials in the water

are responsible for its reducing properties, a conclusion that is supported by work involving the

reduction of Hg(II) to Hg(0) by humic acids (Bloom, unpublished work). The second observation

is that the freezing of water inexplicably, but reproducibly causes the oxidation of As(III) to As(V)

(Figure 2-4-g, i), except in the case of very rapid freezing by immersion in LN2 (Figure 2-4-m, o).

In light of these observations, the following storage regimes are recommended for arsenic in

aqueous solution: 

1. If only total inorganic arsenic plus MMA and DMA are to be determined, the sample should be

stored at 0 to 4°C in polyethylene bottles until analysis. No chemical preservative is needed or

desired and the analysis should be carried out as soon as possible. 

2. If the As(III)/As(V) ratio is to be maintained, the sample must be quick-frozen to -196°C in liquid

nitrogen, and then stored at at least -80°C until analysis. Note that Figure 4-k shows that even

in the case of rapid freezing to -196°C, followed by storage at -18°C, a definite oxidation of

As(III) to As(V) was observed. 

A convenient and safe way to quick-freeze samples is to place 55 ml of sample into a 60-ml

narrow-mouth polyethylene bottle, screw on the cap (which has a 2 mm diameter hole) tightly, and

drop into a Dewar flask full of liquid nitrogen. These bottles have been shown not to crack if less

than 58 ml of water is placed in them, and not to float in the LN2 if more than 50 ml is placed in

them. After returning to the laboratory, the bottles may be placed into a low temperature freezer

until analysis. Note of caution, if a small hole is not placed in the lid of the bottles, which are frozen

in liquid nitrogen, the bottles may explode when removed from the liquid nitrogen. 

Determination of Arsenic Species in Sediments

Two procedures were investigated in the determination of arsenic in sediments. One, a wet-acid

digestion was used to determine total arsenic. The second was a mild, pH-selective leach to

remove various arsenic species intact. 

Total Arsenic. In applying the hot HNO3/H2SO4 digestion to standard sediments and air particulate

matter, good agreement was attained between the established values and the measured values

(Table 2-5). Also, in the case of estuarine and riverine sediments collected in the Puget Sound

area, there was good agreement between X-ray fluorescence spectroscopy and tfiis method (Table

2-6). In either case, all observed arsenic was in the inorganic form. 
2-8

However, when Lake Washington sediment spiked with inorganic as well as organic forms was

analyzed by this method, the following was observed: 

1. All of the MMA was recovered as MMA. 

2. All of the inorganic arsenic was recovered as inorganic arsenic. 

3. None of the DMA was recovered, but an unidentified higher boiling peak was generated. 

This peak is clearly illustrated in Figure 2-5. Even after the above samples were redigested to neardryness (white fumes) in HNO3 plus HClO4, the same results were obtained. Therefore, at this point

we recommend no hydride generation method to determine total arsenic in sediments, though this

may be achieved using either neutron activation analysis or X-ray fluorescence spectroscopy. On

the other hand, since no organic forms have been detected in any natural sediment and since both

MMA and DMA give observable peaks if they are present, it is safe to assume as a general

guideline that if only an inorganic arsenic peak is generated by a given sample, then it probably

represents close to the total arsenic content of the sample. 

Arsenic Speciation of Sediments. Maher (4) has shown that various arsenic species that may be

removed from solids at different pH values. This approach was tested on a sample of spiked Lake

Washington mud, over a wide range of pH using phosphate buffers. The results of these

experiments, shown as arsenic recovered versus pH for all four species, are illustrated in

Figure 2-6. Notice that the maximum recovery of As(III) occurs at about pH = 2.8 and that the

maximum for As(V), MMA and DMA occur at pH >12. From these data, the two convenient buffers

of 0.1 M H3PO4 (pH = 1.5) and Na3PO4 (pH = 12) were chosen to selectively extract the arsenic

species from sediments. Samples extracted with H3PO4. (final pH = 2.3) are analyzed only for

As(III) whereas those extracted with Na3PO4 (final pH = 11.9) are analyzed only for total As, which

gives As(V), MMA and DMA, as As(III) is not extracted at this pH. On untested sediment types it

would be wise to test this relationship to be sure it holds true before instituting an analytical regime.

Recovery of arsenic species from spiked Lake Washington mud is illustrated in Table 2-7. The

calculated spike was added to the mud, which was then aged 14 days at 4°C before analysis. All

analysis were carried out in quintuplicate. The yields are good and within the day-to-day variability

for the respective species. 

The values of the above analysis were then taken as the time zero values, and the mud divided and

stored in one of two ways. Three aliquots each of Lake Washington mud (LWM) and spiked LWM

were placed into polyethylene bottles and frozen at -18°C, while three aliquots were kept

refrigerated at 0 to 4°C. After 30 days these samples were analyzed for arsenic species, the results

of which are shown in Table 2-8. These data indicate that small changes in the concentrations of

the various species may be occurring, with significant decreases (20-30%) in the organic species

being seen. These changes are small enough, however, that if the samples were analyzed as soon

as possible after collection, they should not be of great importance. 

Interstitial Water. Interstitial water is collected from mud by pressure filtration under nitrogen. An

aliquot (~100 g) of mud is placed into a plastic pressure filtration vessel with 1.0 μ acid-cleaned

filter, and tapped down to remove air bubbles. The system is pressurized to 75 psi, and after

discarding the first 1 to 2 ml of filtrate, the interstitial water is collected into a 30-ml polyethylene

bottle under nitrogen. The As(III) stability curve in Figure 2-7 was generated on a sample in contact

with air. Within 5 minutes, the sample had changed from colorless to brown, indicating that Fe(II)
2-9

had oxidized to Fe(III), and precipitated as colloidal Fe(OH)3. If an aliquot of sediment is filtered

under nitrogen and then frozen at -196°C, as for water samples, within 5 to 10 minutes, minimal

changes in the As(III)/As(V) ratio should have taken place. 

Using the above technique, a sample of spiked, Lake Washington sediment was analyzed for

interstitial water arsenic speciation 30 days after spiking with arsenic. This data is presented in

Table 2-9 and shows that the distribution coefficients (Kd) of the various species between the solid

and aqueous phases increase in the following order: DMA<<MMA<As(III)<<As(V). In fact, a sizable

fraction (4.3%) of the DMA is in the interstitial water in a given sample, a fact which is important

considering the intimate interaction of the interstitial water and living creatures. 

Interlaboratory Comparison

An interlaboratory comparison exercise was conducted between Battelle-Northwest (BNW) and

Dr. M.O. Andreae of Florida State University (FSU) to demonstrate the effectiveness of the sample

storage and shipping procedure and verify the accuracy of the anlaytical technique for

determination of arsenic species in fresh water. Three samples were prepared as follows: (1)

Dungeness River water (DRW) was filtered, (2) filtered DRW was spiked with nominally 0.45 μg

L-1 of As (V) and 2 μg L-1 each of DMA and MMA, and (3) coal fly ash, standard reference material

NBS-1633, was leached with DRW then filtered. All solutions were frozen immediately after

preparation in liquid nitrogen then transferred and stored at -80°C. Samples were shipped on dry

ice. Samples were analyzed at BNW and FSU the same week approximately two months after

preparation. The results in Table 2-10 show good agreement between these two laboratories even

for concentrations below 0.1 μg L-1. We believe this interlaboratory exercise has demonstrated that

these storage and shipping procedures are appropriate for freshwater samples and the analytical

method used for arsenic speciation is sensitive and accurate for concentrations of inorganic arsenic

greater than approximately 0.05 and for organic arsenic concentrations greater than 0.2 μg L-1. 

Precision for Sediments and Water

The precision or reproducibility for replicate analyses of arsenic species in field samples is shown

in Table 2-11. Collection of these field samples is described in Section 3 of this report. The

sediment was analyzed for leachable As (III) and As (V). Interstitial water and water from Hyco

Reservoir were also analyzed for As (III) and (V). The results indicate that the relative standard

deviations (RSD) for arsenic (III) and (V) in sediment are approximately 20% while the RSD for

these species in interstitial water and in the water column are approximately 15% and 7%. 

CONCLUSIONS AND RECOMMENDATIONS FOR FURTHER WORK

Arsenic speciation of a variety of materials in the limnological environment is simply and

reproducibly achieved using selective hydride generation/low-temperature trapping techniques in

conjunction with atomic absorption detection. The most difficult problem is the unambiguous

determination of total arsenic in solids by this technique. Other related techniques that might be

investigated include dry ashing, lithium metaborate fusion, and graphite furnace atomic absorption.

An alternate method is to analyze select samples by X-ray fluorescence spectrometry. 
2-10

Figure 2-1. Arsenic Speciation Apparatus: (a) Schematic Diagram, (b) Reaction Vessel, (c)

Quartz Cuvette Burner Tube.
2-11

Figure 2-2. Typical chromatogram of arsenic hydride species. Vertical axis absorbance,

horizontal axis time.
2-12

Figure 2-3. Standard curves, absorbance versus concentration for arsenic hydride species,

atomic absorption detector.
2-13

Figure 2-4a-p. Results of aqueous arsenic species storage tests. Plotted are the percentages

of soluble arsenic species remaining versus storage time.
2-14

Figure 2-4a-p. (continued)
2-15

Figure 2-5. Chromatogram of digested (HNO3/H2SO4) spiked Lake Washington mud. 

Vertical axis absorbance, horizontal axis time. Note absence of DMA peak and presence of

unidentified higher boiling compound.
2-16

Figure 2-6. Arsenic species released from sediments as a function of solution pH. Plot of

arsenic in sediment leached, μg g-1 dry weight basis (DWB), versus pH of leachate.
2-17

Figure 2-7. Plot of the concentration of As(III) and total inorganic arsenic versus storage time in

interstitial water.
2-18

Table 2-1

Optimal Flows and Pressures for Gases

in the Hydride Generation System

Gas

Flow rate

ml • min-1

Pressure

lb • in-2

He 150 10

H2 350 20

Air 180 20

Table 2-2

Reduction Products and Their Boiling Points

of Various Aqueous Arsenic Species

Aqueous form

Reduction

product B.P., °C

As(III), arsenous acid, HAsO2 AsH3 -55

As(V), arsenic acid, H3AsO4 AsH3 -55

MMA, CH3AsO(OH)2 CH3AsH2 2

DMA, (CH3)2AsO(OH) (CH3)2AsH 35.6
2-19

Table 2-3

Replicate Determinations of Total Inorganic

Arsenic in Some Standard Waters

Replicate

Total (inorganic) arsenic, μg•1-1

NASS-1 Seawater NBS River water

1 1.579 81.5

2 1.556 74.5

3 1.591 71.8

4 1.493 79.0

5 1.529 79.3

N5 5

X 1.550 77.2

S 0.040 4.0

RSD 2.6% 5.Z%

Certified 1.65 76.0

± 0.19 7.0

M = number of replicates.

X = mean

S = + one standard deviation

RSD = relative standard deviation

Table 2-4

Precision Data for Three Arsenic Species, Illustrating

The Decrease in Precision with Increasing Boiling

Point of Species. These Samples Were Spiked River

Water Used in Water Storage Tests

Replicate

Arsenic concentrations, ng•1-1

Inorganic

arsenic MMA DMA

N (8-24-83) 3 3 3

X 937 2483 2173

S 44 79 181

RSD 4.7% 3.2% 8.3%

N (9-11-83) 3 4 4

X 800 2342 2393

S 24 165 260

RSD 3.0% 7.0% 10.9%
2-20

Table 2-5

Total Inorganic Arsenic in Standard Sediments by HNO3/H2SO4

Replicate

Total (inorganic) arsenic μg•g-1 dry weight basis

MESS-1

Estuarine

sediment

BCSS-1

Estuarine

sediment

NBS-1646

Estuarine

sediment

NBS-1648

Air

particulate

matter

1 8.9 10.9 9.8 123.0

2 8.8 8.5 10.0 136.0

3 8.8 9.4 9.8 115.0

4 9.6 9.8 8.5 -

5 10.1 10.7 11.0 -

N5 5 5 3

X 9.2 9.9 9.8 125.0

S 0.6 1.0 0.9 11.0

RSD 6.5% 10.1% 9.2% 8.8%

Certified 10.6 11.1 11.6 115.0

+ 1.2 1.4 1.3 10.0
2-21

Table 2-6

Comparison of X-ray Fluorescence Spectroscopy and Hydride

Generation Aa in the Determination of Total Arsenic

Enyironmental Sediments. All Represent Total Inorganic

Arsenic by Hot Acid Digestion Except (*) Slwm,

Which Is the Sum of Species by Leaching

Types of Sediment

Total arsenic, μg•g-1 dry weight basis

XRF Hydride AA

Lake Washington (silt) 14.6 + 0.1 n=3 14.5 + 1.1 n=6

Spiked Lake Washington (silt) 124.1 + 3.4 n=3 120.0 + 7.5 n=5*

BCSS-1, clean estuarine (mud) 11.7 + 0.7 n=3 9.9 + 1.0 n=5

Contaminated Puget Sound (sandy) 108.0 + 24.0 n=3 93.0 + 21.0 n=3

Duwamish River (sand) 8.0 n=1 2.6 n=1

Table 2-7

Recovery of Arsenic Species from Spiked Lake Washington

Mud by Selective Leaching

Arsenic

species

μg•g-1 Arsenic, dry weight basis

Lake

Washington

mud

Spike

added Total recovered

Percent

recovery

As(III) 2.2 + 0.3 5.8 8.2 + 14 103%

As(V) 4.4 + 0.3 9.5 13.5 + 17 96%

MMA <0.8 58.0 51.3 + 6.0 88%

DMA <0.8 54.0 47.0 + 4.2 87%
2-22

Table 2-8

Thirty-day Storage Results for Arsenic Speciation in Sediments

Lake Washington mud

Arsenic

species

μg • g-1 Arsenic, dry weight basis

Initial concentration

Concentrations after 30-day aging

Refrigerated, 0-4C Frozen, -18C

As(III) 2.2 + 0.3 2.2 + 0.4 2.3 + 0.3

As(V) 4.4 + 0.3 5.2 + 0.4 5.4 + 0.4

MMA <0.8 <0.8 <0.8

DMA <0.8 <0.8 <0.8

Spiked Lake Washington mud

Arsenic

species

μg • g-1 Arsenic, dry weight basis

Initial concentration

Concentrations after 30-day aging

Refrigerated, 0-4C Frozen, -18C

As(III) 8.2 + 1.4 7.1 + 2.7 9.9 + 1.3

As(V) 13.5 + 1.7 13.8 + 1.0 16.0 + 0.5

MMA 51.3 + 6.0 39.9 + 1.6 46.2 + 3.5

DMA 47.0 + 4.2 46.5 + 3.2 40.0 + 2.4

Table 2-9

Arsenic Speciation of Spiked Lake Washington Mud

Interstitial Water Kd Values Represent [As (Dry Weight

Sediment]/[As (Insterstitial Water)]

Species

Arsenic concentration μg • g-1

Dry

sediment

Interstitial

water Kd

As(V) 20 <0.002 >10,000

As(III) 5.2 0.014 371

MMA 40 0.11 364

DMA 38 1.72 23
2-23

Table 2-10

Arsenic Speciation Intercomparison Exercise

Sample

μg 

-1

AS (III) As (V) MMA DMW

BNW Andreae BNW Andreae BNW Andreae BNW Andreae

DRW 0.061

+0.004

0.067 0.042

+0.008

0.023 <0.01 0.002 <0.01 0.067

SDRW 0.061

+0.005

0.066 0.468

+0.028

0.421 1.96

+0.11

1.67 1.92

+0.13

1.82

FA 0.052

+0.006

0.031 12.9

+0.2

12.0 <0.01 ND <0.01 ND

Intercomparison exercise results with Meinrat 0. Andreae for arsenic speciation in limnological samples. DRW is

filtered Dungeness River water; SDRW is Dungeness River water spiked with nominally 0.45 μg • 

-1 As (V), and 2 μg

• 

-1 each DMA and MMA. FA is the filtrate of 1000 mg Q-1 NBS coal fly ash leached with DRW.

BNW results are the mean of (3) determinations. ND means not detected. ± = one standard deviation. 
2-24

Table 2-11

Precision of Arsenic Speciation HYCO Reservoir

(February 1984)

Replicate

Sediment As, Sta. 5

μg g-1 dry wt

Interstitial As, Sta. 5

μg L-1

Water co

μ

Total AS (V) AS III) Total As (V) AS (III) Total As 

1 38.33 25.15 13.18 75.8 41.1 34.7 1.222 12 36.61 21.74 14.87 67.1 29.9 37.2 1.082 03 25.27 15.24 10.03 77.2 32.0 45.2 1.186 14 21.28 12.75 8.53 -- -- -- -- -- 

5 29.49 17.26 12.23 -- -- -- -- -- 

6 28.71 16.97 11.74 -- -- -- -- -- 

N 6 6 6 3 3 33 3 

X29.95 18.19 11.76 73.4 34.4 39.0 1.163 1

S 6.53 4.51 2.26 5.5 6.0 5.5 0.073 0

RSD 21.8% 24.8% 19.2% 7.5% 17.4% 14.1% 6.3% 6
2-25

REFERENCES 

1. Braman, R. S., L. L. Johnson, C. C. Foreback, J. M. Ammons and J. L. Bricker. “Separation and

determination of nanogram amounts of inorganic arsenic and methylarsenic compounds.”

Analytical Chemistry 49 (4):621-625 (1977). 

2 Andreae, M. O. “Determination of arsenic species in natural waters.” Analytical Chemistry

49:820, May 1977. 

3. Andreae, M. O. “Methods of Seawater Analysis.” Arsenic (by hydride generation/AAS), pp. 168-

173 Verlag Chemie, Florida, (1983). 

4. Maher, W. A. “Determination of inorganic and methylated arsenic species in marine organisms

and sediments.” Analytica Chemica Acta 126:157-165 (1981). 
APPENDIX I

QUALITY ASSURANCE AND

QUALITY CONTROL GUIDANCE
APPENDIX I

I-3

APPENDIX I

QUALITY ASSURANCE(QA) AND

QUALITY CONTROL (QC) GUIDANCE

I.1 GENERAL QA AND QC CONSIDERATIONS

The primary objective of the specific QA and QC guidance provided in this

document is to ensure that

• Appropriate data quality objectives or requirements are established prior to

sample collection and analysis.

• Samples are collected, processed, and analyzed according to scientifically

valid, cost-effective, standardized procedures.

• The integrity and security of samples and data are maintained at all times.

• Recordkeeping and documentation procedures are adequate to ensure the

traceability of all samples and data from initial sample collection through final

reporting and archiving and to ensure the verifiability and defensibility of

reported results.

• Data quality is assessed, documented, and reported properly.

• Reported results are complete, accurate, and comparable with those from

other similar monitoring programs.

I.2 QA PLAN REQUIREMENTS

To ensure the quality, defensibility, and comparability of the data used to

determine exposure assessments and fish consumption advisories, it is essential

that an effective QA program be developed as part of the overall design for each

monitoring program. The specific QA activities should be documented in a

written QA Project Plan (QAPP) or in a combined Work/QA Plan and should be

implemented strictly throughout all phases of the monitoring program.

The QAPP should follow the guidelines and requirements specified in EPA

Guidance for Quality Assurance Project Plans (EPA QA/G-5) and EPA

Requirements for Quality Assurance Project Plans for Environmental Data (EPA
APPENDIX I

I-4

QA/R-5), where applicable. To obtain the type and quality of environmental data

needed for decision making or a specified end use, the QAPP needs to provide

a project-specific strategy for applying QA and quality control (QC) procedures.

The QAPP should be composed of standardized, recognizable elements that

cover the entire project. These elements should be organized under four general

categories that correspond to the planning, implementation, assessment, and

validation phases of the project. Although project-specific tailoring of the EPA

guidance for developing QA plans is encouraged, all required information must

be included either in full or by reference to appropriate standard operating

procedures (SOPs). The following summarizes the pertinent elements of a QAPP

for each phase of the project.

1. Project Management

a. A historical and scientific perspective of the project including a

description of the problem to be solved or the decision to be made

b. A clear statement of the project goals and the approach to be used and

an overview of the work to be performed and the schedule of

implementation

c. A description of the program organization and personnel roles and

responsibilities, including responsibility for ensuring adherence to the QA

plan

d. Specification of data quality objectives in terms of accuracy, precision,

representativeness, and completeness, for data generated from each

type of measurement system

e. Identification of special training for project personnel

f. A description of the procedure for obtaining approval for substantive

changes in the monitoring program

g. Detailed description of health and safety procedures

2. Measurement and Data Acquisition

a. Detailed descriptions of field sample collection and handling procedures,

including documentation of

• Target species and size (age) class

• Sampling site locations

• Target contaminants

• Sampling times/schedules
APPENDIX I

I-5

• Numbers of samples and sample replication strategy

• Sample collection procedures

• Sample processing procedures, including sample identification,

labeling, preservation, and storage conditions

• Sample shipping procedures

b. A detailed description of chain-of-custody procedures, including specification of standard chain-of-custody forms and clear assignment of field

and laboratory personnel responsibilities for sample custody

c. Detailed descriptions of laboratory procedures for sample receipt,

storage, and preparation, including specification of the kinds of samples

to be prepared for analyses (e.g., composite vs. individual, whole body

vs. fillet, replicates)

d. Detailed descriptions of the analytical methods used for quantitation of

target contaminants and percent lipid determination

e. Detailed descriptions of methods routinely used to assess data

accuracy, precision, and completeness, including

• Internal QC checks using field, reagent, or method blanks; spiked

samples; split samples; QC samples prepared from standard

reference materials; and replicate analyses

• Calibration checks

• Data quality assessments

f. Detailed descriptions of preventive maintenance procedures for

sampling and analysis equipment

g. Detailed descriptions of calibration procedures for all measurement

instruments, including specification of reference materials used for

calibration standards and calibration schedules

h. Detailed descriptions of recordkeeping and documentation procedures,

including requirements for

• Maintaining field and laboratory logs and notebooks

• Use of standard data collection and reporting forms

• Making changes to original records

• Number of significant figures to be recorded for each type of data

• Units of reporting

• Routine procedures to assess the accuracy and completeness of

records
APPENDIX I

I-6

3. Assessment and Oversight

a. Detailed descriptions of data management and reporting procedures,

including requirements for

• Technical reports

• QA and QC reports

• Data coding procedures

• Database specifications

• QA review of reported data

• Data storage and archiving procedures

b. Detailed descriptions of procedures for internal QC performance and/or

systems audits for sampling and analysis programs

c. Detailed descriptions of procedures for external QA performance and/or

systems audits for sampling and analysis programs, including

participation in certified QA proficiency testing or interlaboratory

comparison programs

d. Detailed descriptions of corrective action procedures in both sampling

and analysis programs, including

• Criteria and responsibility for determining the need for corrective

action

• Procedures for ensuring that effective corrective action has been

taken

• Procedures for documenting and reporting corrective actions

4. Data Validation and Usability

a. Provide the criteria to be used in reviewing and validating the data and

for deciding the degree to which each data item has met its quality

specification

b. Describe the process to be used for validating and verifying data,

including the chain of custody for data throughout the project

c. Include detailed descriptions of data analysis procedures, including

• Statistical treatment of data

• Data summary formats (e.g., plots, tables)

d. Precisely define and interpret how validation issues differ from

verification issues
APPENDIX I

I-7

Guidance for addressing each of the QA or QC elements outlined above, including

a list of recommended standard reference materials and external QA or

interlaboratory comparison programs for the analyses of target analytes, is

incorporated in the appropriate sections of this guidance document. The EPA

guidance and requirements documents (EPA QA/G-5 and EPA QA.R-5) should

be referenced for more detailed discussions of the elements to be included in the

QA plan (available at http://es.epa.gov/ncerqa/qaqa_docs.html).
APPENDIX J

RECOMMENDED PROCEDURES FOR PREPARING

WHOLE FISH COMPOSITE HOMOGENATE SAMPLES
APPENDIX J

J-3

APPENDIX J

RECOMMENDED PROCEDURES FOR PREPARING WHOLE

FISH COMPOSITE HOMOGENATE SAMPLES

J.1 GENERAL GUIDELINES

Laboratory processing to prepare whole fish composite samples (diagrammed in

Figure J-1) involves

• Inspecting individual fish for foreign material on the surface and rinsing if

necessary

• Weighing individual fish

• Examining each fish for morphological abnormalities (optional)

• Removing scales or otoliths for age determination (optional)

• Determining the sex of each fish (optional)

• Preparing individual whole fish homogenates

• Preparing a composite whole fish homogenate.

Whole fish should be shipped on wet or blue ice from the field to the sample

processing laboratory if next-day delivery is assured. Fish samples arriving in this

manner (chilled but not frozen) should be weighed, scales and/or otoliths

removed, and the sex of each fish determined within 48 hours of sample

collection. The grinding/homogenization procedure may be carried out more

easily and efficiently if the sample has been frozen previously (Stober, 1991).

Therefore, the samples should then be frozen (-20 C) in the laboratory prior to

being homogenized.

If the fish samples arrive frozen (i.e., on dry ice) at the sample processing

laboratory, precautions should be taken during weighing, removal of scales and/or

otoliths, and sex determination to ensure that any liquid formed in thawing

remains with the sample. Note: The liquid will contain target analyte

contaminants and lipid material that should be included in the sample for analysis.

The thawed or partially thawed whole fish should then be homogenized

individually, and equal weights of each homogenate should be combined to form

the composite sample. Individual homogenates and/or composite homogenates

may be frozen; however, frozen individual homogenates must be rehomogenized

before compositing, and frozen composite homogenates must be rehomogenized

before aliquotting for analysis. The maximum holding time from sample collection

to analysis for mercury is 28 days at -20 C; for all other analytes, the holding

time is 1 year at -20 C (Stober, 1991). Recommended container materials,
APPENDIX J

J-4

Composite equal weights (g) of 

homogenized fillet tissues from the 

selected number of fish (200-g)

Seal and label (200-g) composite 

homogenate in appropriate container(s) 

and store at ≤-20 °C until analysis (see 

Table 7-1 for recommended container 

materials and holding times).

Log in fish samples using COC procedures

Unwrap and inspect individual fish

Weigh individual fish

Remove and archive scales and/or otoliths for age determination (optional)

Determine sex (optional); note morphological abnormalities (optional)

Save remainder of fillet

homogenate from each fish

Seal and label individual fillet 

homogenates in appropriate 

container(s) and archive at 

≤-20 °C (see Table 7-1 for 

recommended container 

materials and holding times).

Remove scales from all scaled fish Remove skin from scaleless fish (e.g., catfish) 

COC = Chain of custody.

Fillet fish

Weigh fillets (g)

Homogenize fillets

Divide homogenized sample into quarters, mix opposite

quarters, and then mix halves (3 times)

Optional

Figure J-1. Laboratory sample preparation and handling for whole fish

composite homogenate samples.
APPENDIX J

J-5

preservation temperatures, and holding times are given in Table J-1. Note:

Holding times in Table J-1 are maximum times recommended for holding samples

from the time they are received at the laboratory until they are analyzed. These

holding times are based on guidance that is sometimes administrative rather than

technical in nature; there are no promulgated holding time criteria for tissues (U.S.

EPA, 1995b). If states choose to use longer holding times, they must

demonstrate and document the stability of the target analyte residues over the

extended holding times.

J.2 SAMPLE PROCESSING PROCEDURES

Fish sample processing procedures are discussed in more detail in the sections

below. Each time custody of a sample or set of samples is transferred from one

person to another during processing, the Personal Custody Record of the chainof-custody (COC) form that originated in the field (Figure 6-8) must be completed

and signed by both parties so that possession and location of the samples can be

traced at all times (see Section 7.1). As each sample processing procedure is

performed, it should be documented directly in a bound laboratory notebook or

on standard forms that can be taped or pasted into the notebook. The use of a

standard form is recommended to ensure consistency and completeness of the

record. Several existing programs have developed forms similar to the sample

processing record for whole fish composite samples shown in Figure J-2. 

J.2.1 Sample Inspection

Individual fish received for filleting should be unwrapped and inspected carefully

to ensure that they have not been compromised in any way (i.e., not properly

preserved during shipment). Any specimen deemed unsuitable for further

processing and analysis should be discarded and identified on the sample

processing record.

J.2.2 Sample Weighing

A wet weight should be determined for each fish. All samples should be weighed

on balances that are properly calibrated and of adequate accuracy and precision

to meet program data quality objectives. Balance calibration should be checked

at the beginning and end of each weighing session and after every 20 weighings

in a weighing session. 

Fish shipped on wet or blue ice should be weighed directly on a foil-lined balance

tray. To prevent cross contamination between individual fish, the foil lining should

be replaced after each weighing. Frozen fish (i.e., those shipped on dry ice)

should be weighed in clean, tared, noncontaminating containers if they will thaw

before the weighing can be completed. Liquid from the thawed sample must be
APPENDIX J

J-6

Table J-1. Recommendations for Container Materials, Preservation, and Holding

Times for Fish, Shellfish, and Turtle Tissues from Receipt at Sample Processing

Laboratory to Analysis

Analyte Matrix

Sample

container

Storage

Preservation Holding timea

Mercury Tissue (whole specimens,

homogenates)

Plastic,

borosilicate

glass, quartz,

and PTFE

Freeze at <-20 C 28 daysb

Other metals Tissue (whole specimens,

homogenates)

Plastic,

borosilicate

glass, quartz,

and PTFE

Freeze at <-20 C 6 monthsc

Organics Tissue (whole specimens,

homogenates)

Borosilicate

glass, quartz,

PTFE, and

aluminum foil

Freeze at <-20 C 1 yeard

Metals and

organics

Tissue (whole specimens,

homogenates)

Borosilicate

glass, quartz,

and PTFE

Freeze at <-20 C 28 days

(mercury);

6 months (for

other metals);

and 1 year (for

organics) 

Lipids Tissue (whole specimens,

homogenates)

Plastic,

borosilicate

glass, quartz,

PTFE

Freeze at <-20 C 1 year

PTFE = Polytetrafluoroethylene for Teflon.

a Maximum holding times recommended by U.S. EPA (1995b). b This maximum holding time is also recommended by the Puget Sound Estuary Program (1990). The

California Department of Fish and Game (1990) and the USGS National Water Quality Assessment Program

(Crawford and Luoma, 1993) recommend a maximum holding time of 6 months for all metals, including

mercury. c This maximum holding time is also recommended by the California Department of Fish and Game (1990),

the 301(h) monitoring program (U.S. EPA, 1986), and the USGS National Water Quality Assessment

Program (Crawford and Luoma, 1993). The Puget Sound Estuary Program (1990) recommends a maximum

holding time of 2 years. d This maximum holding time is also recommended by the Puget Sound Estuary Program (1990). The

California Department of Fish and Game (1990) and the USGS National Water Quality Assessment Program

(Crawford and Luoma, 1993) recommend a more conservative maximum holding time of 6 months. EPA

(1995a) recommends a maximum holding time of 1 year at -10 C for dioxins and dibenzofurans.
APPENDIX J

J-7

Figure J-2. Example of a sample processing record for fish contaminant monitoring

program—whole fish composites.
APPENDIX J

J-8

kept in the container as part of the sample because it will contain lipid material

that has separated from the tissue (Stober, 1991).

All weights should be recorded to the nearest gram on the sample processing

record and/or in the laboratory notebook.

J.2.3 Age Determination

Age provides a good indication of the duration of exposure to pollutants (Versar,

1982). A few scales or otoliths (Jearld, 1983) should be removed from each fish

and delivered to a fisheries biologist for age determination. For most warm water

inland gamefish, 5 to 10 scales should be removed from below the lateral line and

behind the pectoral fin. On softrayed fish such as trout and salmon, the scales

should be taken just above the lateral line (WDNR, 1988). For catfish and other

scaleless fish, the pectoral fin spines should be clipped and saved (Versar, 1982).

The scales, spines, or otoliths may be stored by sealing them in small envelopes

(such as coin envelopes) or plastic bags labeled with, and cross-referenced by,

the identification number assigned to the tissue specimen (Versar, 1982).

Removal of scales, spines, or otoliths from each fish should be noted (by a check

mark) on the sample processing record.

J.2.4 Sex Determination (Optional)

To determine the sex of a fish, an incision should be made on the ventral surface

of the body from a point immediately anterior to the anus toward the head to a

point immediately posterior to the pelvic fins. If necessary, a second incision

should be made on the left side of the fish from the initial point of the first incision

toward the dorsal fin. The resulting flap should be folded back to observe the

gonads. Ovaries appear whitish to greenish to golden brown and have a granular

texture. Testes appear creamy white and have a smooth texture (Texas Water

Commission, 1990). The sex of each fish should be recorded on the sample

processing record.

J.2.5 Assessment of Morphological Abnormalities (Optional)

Assessment of gross morphological abnormalities in finfish is optional. This

assessment may be conducted in the field (see Section 6.3.1.5) or during initial

inspection at the central processing laboratory prior to filleting. States interested

in documenting morphological abnormalities should consult Sinderman (1983) and

review recommended protocols for fish pathology studies used in the Puget

Sound Estuary Program (1990).

J.2.6 Preparation of Individual Homogenates

To ensure even distribution of contaminants throughout tissue samples, whole fish

must be ground and homogenized prior to analyses.
APPENDIX J

J-9

Smaller whole fish may be ground in a hand crank meat grinder (fish < 300 g) or

a food processor (fish 300-1,000 g). Larger (>1,000 g) fish may be cut into

2.5-cm cubes with a food service band saw and then ground in either a small or

large homogenizer. To avoid contamination by metals, grinders and homogenizers used to grind and blend tissue should have tantalum or titanium blades

and/or probes. Stainless steel blades and probes have been found to be a

potential source of nickel and chromium contamination (due to abrasion at high

speeds) and should be avoided. 

Grinding and homogenization of biological tissue, especially skin from whole fish

samples, is easier when the tissue is partially frozen (Stober, 1991). Chilling the

grinder/homogenizer briefly with a few chips of dry ice will reduce the tendency

of the tissue to stick to the grinder.

The ground sample should be divided into quarters, opposite quarters mixed

together by hand, and the two halves mixed back together. The grinding,

quartering, and hand mixing should be repeated two more times. If chunks of

tissue are present at this point, the grinding/homogenizing should be repeated.

No chunks of tissue should remain because these may not be extracted or

digested efficiently. If the sample is to be analyzed for metals only, the ground

tissue may be mixed by hand in a polyethylene bag (Stober, 1991). Homogenization of each individual fish should be noted on the sample processing record.

At this time, individual whole fish homogenates may be either composited or

frozen and stored at -20 C in cleaned containers that are noncontaminating for

the analyses to be performed (see Table J-1).

J.2.7 Preparation of Composite Homogenates

Composite homogenates should be prepared from equal weights of individual

homogenates. If individual whole fish homogenates have been frozen, they

should be thawed partially and rehomogenized prior to compositing. Any

associated liquid should be maintained as a part of the sample. The weight of

each individual homogenate that is used in the composite homogenate should be

recorded, to the nearest gram, on the sample processing record.

Each composite homogenate should be blended by dividing it into quarters,

mixing opposite quarters together by hand, and mixing the two halves together.

The quartering and mixing should be repeated at least two more times. If the

sample is to be analyzed only for metals, the composite homogenate may be

mixed by hand in a polyethylene bag (Stober, 1991). At this time, the composite

homogenate may be processed for analysis or frozen and stored at -20 C (see

Table J-1).

The remainder of each individual homogenate should be archived at -20 C with

the designation "Archive" and the expiration date recorded on the sample label.

The location of the archived samples should be indicated on the sample

processing record under "Notes."
APPENDIX J

J-10

It is essential that the weights of individual homogenates yield a composite

homogenate of adequate size to perform all necessary analyses. Weights of

individual homogenates required for a composite homogenate, based on the

number of fish per composite and the weight of composite homogenate

recommended for analyses of all screening study target analytes (see Table 4-1),

are given in Table J-2. The total composite weight required for intensive studies

may be less than in screening studies if the number of target analytes is reduced

significantly.

The recommended sample size of 200 g for screening studies is intended to

provide sufficient sample material to (1) analyze for all recommended target

analytes (see Table 4-1) at appropriate detection limits, (2) meet minimum QA

and QC requirements for the analyses of replicate, matrix spike, and duplicate

matrix spike samples (see Section 8.3.3.4), and (3) allow for reanalysis if the QA

and QC control limits are not met or if the sample is lost. However, sample size

requirements may vary among laboratories and the analytical methods used.

Therefore, it is the responsibility of each program manager to consult with the

analytical laboratory supervisor to determine the actual weights of composite

homogenates required to analyze for all selected target analytes at appropriate

detection limits.

J.3 REFERENCES

California Department of Fish and Game. 1990. Laboratory Quality Assurance

Program Plan. Environmental Services Division, Sacramento, CA.

Crawford, J.K., and S.N. Luoma. 1993. Guidelines for Studies of Contaminants

in Biological Tissues for the National Water-Quality Assessment Program.

USGS Open-File Report 92-494. U.S. Geological Survey, Lemoyne, PA.

Jearld, A. 1983. Age determination. pp. 301-324. In: Fisheries Techniques.

L.A. Nielsen and D. Johnson (eds.). American Fisheries Society, Bethesda,

MD.

Puget Sound Estuary Program. 1990 (revised). Recommended protocols for fish

pathology studies in Puget Sound. Prepared by PTI Environmental Services,

Bellevue, WA. In: Recommended Protocols and Guidelines for Measuring

Selected Environmental Variables in Puget Sound. Region 10, U.S.

Environmental Protection Agency, Seattle, WA. (Looseleaf)

Sinderman, C. J. 1983. An examination of some relationships between pollution

and disease. Rapp. P. V. Reun. Cons. Int. Explor. Mer. 182:37-43.

Stober, Q. J. 1991. Guidelines for Fish Sampling and Tissue Preparation for

Bioaccumulative Contaminants. Environmental Services Division, Region 4,

U.S. Environmental Protection Agency, Athens, GA.
APPENDIX J

J-11

Texas Water Commission. 1990. Texas Tissue Sampling Guidelines. Texas

Water Commission, Austin, TX.

U.S. EPA (U.S. Environmental Protection Agency). 1986. Bioaccumulation

Monitoring Guidance: 4. Analytical Methods for U.S. EPA Priority Pollutants

and 301(h) Pesticides in Tissues from Marine and Estuarine Organisms.

EPA-503/6-90-002. Office of Marine and Estuarine Protection, Washington,

DC.

U.S. EPA (U.S. Environmental Protection Agency). 1995a. Method 1613b.

Tetra- through Octa-Chlorinated Dioxins and Furans by Isotope Dilution

HRGC/HRMS. Final Draft. Office of Water, Office of Science and

Technology, Washington, DC.

U.S. EPA (Environmental Protection Agency). 1995b. QA/QC Guidance for

Sampling and Analysis of Sediments, Water, and Tissues for Dredged

Material Evaluations—Chemical Evaluations. EPA 823-B-95-001. Office of

Water, Washington, DC, and Department of the Army, U.S. Army Corps of

Engineers, Washington, DC.

Versar, Inc. 1982. Sampling Protocols for Collecting Surface Water, Bed

Sediment, Bivalves and Fish for Priority Pollutant Analysis--Final Draft Report.

EPA Contract 68-01-6195. Prepared for U.S. EPA Office of Water

Regulations and Standards. Versar, Inc., Springfield, VA.

WDNR (Wisconsin Department of Natural Resources). 1988. Fish Contaminant

Monitoring Program—Field and Laboratory Guidelines (1005.1). Madison,

WI.
APPENDIX K

GENERAL PROCEDURES FOR REMOVING

EDIBLE TISSUES FROM FRESHWATER TURTLES
APPENDIX K

K-3

APPENDIX K

GENERAL PROCEDURES FOR REMOVING EDIBLE TISSUES

FROM FRESHWATER TURTLES

1. Turtles brought to the processing laboratory on wet, blue, or dry ice should be

placed in a freezer for a minimum of 48 hours prior to resection. Profound

hypothermia can be employed to induce death (Frye, 1994) Decapitation of

alert animals is not recommended because there is evidence that decapitation

does not produce instantaneous loss of consciousness (Frye, 1994).

2. The turtle should be placed on its back with the plastron (ventral plate) facing

upward. The carapace and plastron are joined by a bony bridge on each side

of the body extending between the fore and hindlimbs (Figure K-1). Using a

bone shears, pliers, or sharp knife, break away the two sides of the carapace

from the plastron between the fore and hind legs on each side of the body.

3. Remove the plastron to view the interior of the body cavity. At this point,

muscle tissue from the forelimbs, hindlimbs, tail (posterior to the anus), and

neck can be resected from the body. The muscle tissue should be skinned

and the bones should be removed prior to homogenization of the muscle

tissue. Typically, the muscle tissue is the primary tissue consumed, and turtle

meat sold in local markets usually contains lean meat and bones only (Liner,

1978).

Dietary and culinary habits concerning which turtle tissues are edible,

however, differ greatly among various populations. In some populations, the

liver, heart, eggs, fatty deposits, and skin are also used (Liner, 1978).

Therefore only general information on the types of turtle tissues most

frequently considered edible can be presented here. State staff familiar with

the dietary and culinary habits of the turtle-consuming populations within their

jurisdictions are the best judge of which edible tissues should be included as

part of the tissue samples used to assess the health risks to the turtleconsuming public.

4. Several of the tissue types that are considered edible include the fatty

deposits found in various parts of the body, the heart, liver (usually with the

gall bladder removed), and the eggs (if the specimen is a female). These

edible tissues are shown in Figure K-2.
APPENDIX K

K-4

Source: Ashley, 1962.

Figure K-1.
APPENDIX K

K-5

Source: Ashley, 1962.

Figure K-2.
APPENDIX K

K-6

• Masses of yellowish-green fatty deposits may be removed from above the

forelimbs and from above and in front of the hindlimbs. Fatty deposits can

also be found at the base of the neck near the point where the neck enters

the body cavity. 

• The centrally located heart is positioned anterior to the liver.

• The large brownish liver is the predominant tissue in the body cavity and

is an edible tissue eaten by some populations. Note: The small greenishcolored gall bladder lies on the dorsal side of the right lobe of the liver (not

visible unless the liver is lifted upward and turned over). The gall bladder

is usually removed and discarded by consumers because of its acrid taste

(Liner, 1978). 

• If the turtle specimen is a female, ovaries containing bright yellow-colored

spherical eggs of varying sizes are located posterior to the liver and lie

against the dorsal body wall. 

Note: The fatty deposits, liver tissue, and eggs are highly lipophilic tissues and

have been shown to accumulate chemical contaminants at concentrations 10 to

more than 100 times the concentrations reported from muscle tissue (Bryan et al.,

1987; Hebert et al., 1993; Olafsson et al., 1983, 1987; Ryan et al., 1986; Stone

et al., 1980). States may wish to resect the fatty tissues, liver, heart, and eggs for

inclusion in the turtle muscle tissue sample to obtain a conservative estimate of

the concentration to which the turtle-consuming public would be exposed.

Alternatively, states may want to retain these tissues for individual analysis.

Some states already advise their residents who consume turtles to remove all

fatty tissues (Minnesota Department of Health, 1994; New York State Department

of Health, 1994) and not to consume the liver and eggs (New York State

Department of Health, 1994). These cleaning procedures are recommended as

a risk-reducing strategy.

REFERENCES

Ashley, L.M. 1962. Laboratory Anatomy of the Turtle. W.C. Brown Company

Publishers, Dubuque, IA.

Bryan, A.M., P.G. Olafsson, and W.B. Stone. 1987. Disposition of low and high

environmental concentrations of PCBs in snapping turtle tissues. Bull.

Environ. Contam. Toxicol. 38:1000-1005.

Frye, F.L. 1994. Reptile Clinician’s Handbook: A Compact Clinical and Surgical

Reference. Krieger Publishing Company, Malabar, FL.
APPENDIX K

K-7

Hebert, C.E., V. Glooschenko, G.D. Haffner, and R. Lazar. 1993. Organic

contaminants in snapping turtle (Chelydra serpentina) populations from

Southern Ontario, Canada. Arch. Environ. Contam. Toxicol. 24:35-43.

Liner, E.A. 1978. A Herpetological Cookbook: How to Cook Amphibians and Reptiles.

Privately printed, Houma, LA.

Minnesota Department of Health. 1994. Minnesota Fish Consumption Advisory.

Minneapolis, MN.

New York State Department of Health. 1994. Health Advisory-Chemicals in Sportfish

and Game 1994-1995. #40820042. Division of Environmental Health

Assessment, Albany, NY.

Olafsson, P.G., A.M. Bryan, B. Bush, and W. Stone. 1983. Snapping turtles—A

biological screen for PCBs. Chemosphere 12 (11/12):1525-1532.

Olafsson, P.G., A.M. Bryan, and W. Stone. 1987. PCB congener specific analysis: A

critical evaluation of toxic levels in biota. Chemosphere 16 (10-12):2585-2593.

Ryan, J.J., P.Y. Lau, and J.A. Hardy. 1986. 2,3,7,8, Tetrachlorodibenzo-p-dioxin and

related dioxans and furans in snapping turtle (Chelydra serpentina) tissues

from the upper St. Lawrence River. Chemosphere 15 (5):537-548.

Stone, W.B., E. Kiviat, and S.A. Butkas. 1980. Toxicants in snapping turtles. New York

Fish and Game Journal 27 (1):39-50.
APPENDIX L

GENERAL PROCEDURES FOR REMOVING

EDIBLE TISSUES FROM SHELLFISH

Source: UNC Sea Grant Publication. 1988. UNC-SG-88.02. The Water Resources

Institute, North Carolina State University, Raleigh, NC
APPENDIX L

L-3

Source: UNC Sea Grant. 1988. Publication UNC-SG-88-02. The Water Resources

Research Institute, North Carolina State University, Raleigh, NC.
APPENDIX L

L-4
APPENDIX L

L-5
APPENDIX L

L-6
APPENDIX L

L-7
APPENDIX L

L-8
APPENDIX M

SOURCES OF REFERENCE 

MATERIALS AND STANDARDS
APPENDIX M

M-3

APPENDIX M

SOURCES OF REFERENCE MATERIALS AND STANDARDS

M.1 SOURCES OF REFERENCE MATERIALS

Analytical reference materials for priority pollutants and related compounds are

currently produced for: organic quality control samples; organic solution

standards; organic neat standards; inorganic quality control standards; and solid

matrix quality control standards as listed below.

Note: Mention of trade names or commercial products does not constiitute

endorsement or recommendation for use. Identification of retailers of

these products does not constitute their endorsement.

M.2 RETAILERS OF ORGANIC QUALITY CONTROL SAMPLES

Accustandard

125 Market Street

New Haven, CT 06513

Tel: 203-786-5290

FAX: 203-786-5287

Contact: Mike Bolgar

Aldrich Chemical Company, Inc.

940 West Saint Paul Avenue

Milwaukee, WI 53233

Tel: 414-273-3850

FAX: 800-962-9591

Analytical Products Group

2730 Washington Boulevard

Belpre, OH 45714

Tel: 704-423-4200

FAX: 704-423-5588

1-800-272-4442

Contact: Tom Coyner/

Melissa McNamara

Crescent Chemical Corporation

1324 Motor Parkway

Hauppauge, NY 11788

Tel: 516-348-0333

FAX: 516-348-0913

Contact: Fran Seiss

Environmental Research Associates

5540 Marshall Street

Arvada, CO 80002

Tel: 303-431-8454

FAX: 303-421-0159

Contact: Mark Carter

NSI Environmental Solutions, Inc.

P. O. Box 12313

2 Triangle Drive

Research Triangle Park, NC 27709

Tel: 1-800-234-7837 or

1-919-549-8980

FAX: 1-919-544-0334

Restek Corporation

110 Benner Circle

Bellefonte, PA 16823

Tel: 814-353-1300

FAX: 814-353-1309

Contact: Eric Steindle
APPENDIX M

M-4

Supelco

Supelco Park

Bellefonte, PA 16823-0048

Tel: 800-247-6628 or 

814-359-3441

FAX: 814-359-3044

Ultra Scientific

250 Smith Street

North Kingstown, RI 02852

Tel: 401-294-9400

FAX: 401-295-2330

M.3 RETAILERS OF ORGANIC SOLUTION STANDARDS

Absolute Standards, Inc.

P.O. Box 5585

Hamden, NJ 06518-0585

Tel: 800-368-1131

FAX: 800-410-2577

Contact: Jack Criscio

Accustandard

125 Market St.

New Haven, CT 06513

Tel: 203-786-5290

FAX: 203-786-5287

Contact: Mike Bolgar

Alameda Chemical and Scientific

922 East Southern Pacific Drive

Phoenix, AZ 85034

Tel: 602-256-7044

FAX: 602-256-6566

Contact: Jim Stauffer

Cambridge Isotope Laboratories

50 Frontage Road

Andover, MA 01801-5413

Tel: 800-322-1174 or 

978-749-8000

FAX: 978-749-2768

Contact: Jim Grim

NSI Environmental Solutions, Inc.

P.O. Box 12313

2 Triangle Drive

Research Triangle Park, NC 27709

Tel: 800-234-7837 or 919-549-8980

FAX: 919-544-0334

Contact: Zora Bunn

Research Technology Corporation

2931 Soldier Springs Road

P. O. Box 1346

Laramie, WY 82070

Tel: 307-742-6343

FAX: 307-745-7936

Contact: Robert Rucinski

M.4 RETAILERS OF NEAT ORGANIC STANDARDS

Accustandard

125 M arket St.

New Haven, CT 06513

Tel: 203-786-5290

FAX: 203-786-5287

Contact: Mike Bolgar

NSI Environmental Solutions, Inc.

P. O. Box 12313

2 Triangle Drive

Research Triangle Park, NC 27709

Tel: 1-800-234-7837 or

1-919-549-8980

FAX: 1-919-544-0334
APPENDIX M

M-5

M.5 RETAILERS OF INORGANIC QUALITY CONTROL SAMPLES

SPEX Industries, Inc.

203 Norcross Ave.

Metuchen, NJ 08840

Tel: 732-549-7144 or 

1-800-522-7739

FAX: 732-603-9647

NSI Environmental Solutions, Inc.

P. O. Box 12313

2 Triangle Drive

Research Triangle Park, NC 27709

Tel: 1-800-234-7837 or

1-919-549-8980

FAX: 1-919-544-0334

M.6 RETAILERS OF SOLID MATRIX QUALITY CONTROL SAMPLES

Fisher Scientific

711 Forbes Avenue (Corporate

address)

Pittsburgh, PA 15219

Tel: 1-800-227-6701

FAX: 1-800-926-1166

NSI Environmental Solutions, Inc.

P. O. Box 12313

2 Triangle Drive

Research Triangle Park, NC 27709

Tel: 1-800-234-7837 or

1-919-549-8980

FAX: 1-919-544-0334

M.7 RECOMMENDED PUBLICATIONS ON CERTIFIED STANDARDS AND REFERENCE

MATERIALS

• Standard and Reference Materials for Marine Science (NOAA, 1992). 

Available from

Dr. Adrianna Cantillo

National Ocean Service

National Oceanic and Atmospheric Administration

U.S. Department of Commerce

1305 East West Highway

Silver Spring, MD 20910

This catalog lists approximately 2,000 reference materials from 16

producers and includes information on their use, sources, matrix type,

analyte concentrations, proper use, availability, and costs. Reference

materials are categorized as follows: ashes, gases, instrumental

performance, oils, physical properties, rocks, sediments, sludges, tissues,

and waters. This catalog has been published independently by both NOAA

and IOC/UNEP and is available in electronic form from the Office of Ocean

Resources, Conservation, and Assessment, NOAA/NOS.

• Biological and Environmental Reference Materials for Trace Elements,

Nuclides and Organic Microcontaminants (Toro et al., 1990). Available

from

Dr. R.M. Parr

Section of Nutritional and Health-Related Environmental Studies

International Atomic Energy Agency

P.O. Box 100

A-1400 Vienna, Austria
APPENDIX M

M-6

This report contains approximately 2,700 analyte values for 117 analytes in

116 biological and 77 nonbiological environmental reference materials from

more than 20 sources. Additional information on cost, sample size

available, and minimum amount of material recommended for analysis is

also provided.

M.8 REFERENCES

NOAA (National Oceanic and Atmospheric Administration). 1992. Standard

and Reference Materials for Marine Science. Third Edition. U.S.

Department of Commerce, Rockville, MD.

Toro, E. C., R. M. Parr, and S. A. Clements. 1990. Biological and

Environmental Reference Materials for Trace Elements, Nuclides and

Organic Microcontaminants: A Survey. IAEA/RL/128(Rev. 1). 

International Atomic Energy Agency, Vienna, Austria.
APPENDIX N

STATISTICAL METHODS FOR COMPARING SAMPLES:

SPATIAL AND TEMPORAL CONSIDERATIONS
APPENDIX N

N-3

APPENDIX N

STATISTICAL METHODS FOR COMPARING SAMPLES:

SPATIAL AND TEMPORAL CONSIDERATIONS

The primary objective of Tier 2 intensive studies is to assess the magnitude and

geographic extent of contamination in selected target species by determining

whether the mean contaminant concentration exceeds the screening value (SV)

for any target analyte. Secondary objectives of intensive studies may include

defining the geographical region where fish contaminant concentrations exceed

screening values (SVs), identifying geographic distribution of contaminant

concentrations, and, in conjunction with historical or future data collection,

assessing changes in fish contaminant concentrations over time. This appendix

discusses some of the statistical methods that may be used to compare fish

contaminant levels measured at different locations or over time.

The recommended statistical approach for comparing replicated contaminant

measurements between two or more groups is outlined below and in Figure N-1.

For each type of test, several options are provided, each of which may be

appropriate in specific cases. State staff should consult a statistician as to the

specific statistical tests to use for a particular data set.

Statistical tests of significant differences between means (or other measures of

central tendency) can be divided into parametric and nonparametric types.

Parametric tests assume that the contaminant concentrations in the population

being sampled are normally distributed and that the population variances in the

groups being tested are not significantly different from each other (Gilbert, 1987).

If either of these assumptions is violated, a nonparametric test may be more

appropriate. However, nonparametric tests should be used only when necessary

because the power of parametric tests generally is greater than the power of

nonparametric tests when the assumptions of the parametric test have been met

(Sokal and Rohlf, 1981).

Because the populations of many environmental measurements are not normally

distributed, logarithmic transformation is often performed on the sampled data

(Gilbert, 1987). However, transformation may not be appropriate in all cases. If

the data are sampled from a population that is normally distributed, then there is

no need for transformation (Figure N-1).

If the assumptions of normality and equality of variance are met, parametric tests

of significant differences between means, such as the one-way Analysis of
APPENDIX N

N-4

Figure N-1. Statistical approach to testing for significant differences between different

groups of contaminant monitoring data.
APPENDIX N

N-5

Variance (ANOVA) and the t-test, should be performed. If three or more groups

are compared using the ANOVA that results in a significant difference, the

difference in mean concentrations between two group means can be further

investigated using a multiple comparison test (Figure N-1). These tests indicate

which specific means are significantly different from each other, rather than just

indicating that one or more means are different, as the ANOVA does.

If the underlying assumptions for parametric testing are not met, nonparametric

tests of significance can be employed. Nonparametric tests of significant differences in central tendencies are often performed on transformed data, that is, the

ranks. Multiple comparison tests comparable to those used for parametric data

sets are not available for nonparametric data sets. For data sets including three

or more groups, a series of two-sample tests can be performed that can yield

similar information to that derived from multiple comparison tests.

Because the concentrations of contaminants, particularly nonpolar organics, are

often correlated with the percentage of lipid in a tissue sample (see Section 8.1.2),

contaminant data are often normalized to the lipid concentration before statistical

analyses are performed. This procedure can, in some instances, improve the

power of the statistical tests. States wishing to examine the relationship between

contaminant concentrations and percentage of lipid should refer to Hebert and

Keenleyside (1995) for a discussion of the possible statistical approaches.

Intensive studies may include the collection of fish contaminant data from several

locations within a region of interest or for multiple time periods (e.g., seasons or

years) from a single location, or a combination of both. Data from intensive

studies such as these may be used to perform spatial (i.e., between stations) or

temporal (i.e., over time) analyses. It should be noted that these types of

analyses, if performed, are performed in addition to the statistical comparisons of

mean target analyte concentrations with SVs described in Section 6.1.2.7. It is

only the latter type of comparison that should be used to make decisions

regarding the necessity of performing risk assessments and the issuance of fish

consumption advisories. Spatial and temporal comparisons of contaminant data,

however, may yield important information about the variability of target analyte

concentrations in specific populations of a particular target species.

N.1 SPATIAL COMPARISON OF STATIONS

Intensive studies also may involve the collection of contaminant data from multiple

stations within a waterbody of interest. The stations could be located in different

lakes within a single drainage basin, upstream and downstream of a point source

of concern along a single river, or randomly located within a single waterbody if

an estimate of random spatial variability is desired. The use of an example will

serve to illustrate how a spatial analysis of contaminant data might be performed.

In this example, a state has determined from a screening study on a river that

cadmium is present in a target species at 20 ppm, which is two times the state

selected SV of 10 ppm. An intensive survey was undertaken in which eight
APPENDIX N

N-6

Table N-1. Hypothetical Cadmium Concentrations (ppm) in

Target Species A at Three River Locations

Replicate samples Station 1 Station 2 Station 3

1 20 28 33

2 18 27 30

3 25 34 30

4 22 28 28

5 21 30 20

6 22 29 39

7 23 30 31

8 21 29 30

Mean 21.5 29.4 31.3

Standard deviation 2.07 2.13 3.45

p-Value for t-test with SV <0.001 <0.001 <0.001

p-Value for W test 0.97 0.83 0.78

p-Value for Levene’s test 0.52

p-Value for ANOVA <0.0001

p-Value for Duncan’s-1 vs. 2 <0.0001

p-Value for Duncan’s-1 vs. 3 >0.0001

p-Value for Duncan’s-2 vs. 3 0.17

samples were collected from three locations on the river of potential concern and

analyzed for cadmium. The results of the analyses for each location and the

statistical comparisons between the three groups are presented in Table N-1.

The mean cadmium concentration at each of three locations was more than twice

the selected SV of 10 ppm (Table N-1). The most important statistical test, as

indicated in Section 6.1.2.7, is a comparison of the mean target analyte concentration for each location with the appropriate SV for that target analyte using a ttest. These tests must be performed before any analysis of spatial trends is

performed. The results of the t-tests indicate that each of the three mean tissue

concentrations is significantly greater than the SV (Table N-1). By itself, these

results indicate that a risk assessment is warranted.

A general statistical flowchart for comparing contaminant concentration data from

several stations is presented in Figure N-1. The cadmium data in Table N-1 may

be additionally analyzed using the tests in Figure N-1. All of the statistical tests

in Figure N-1 can be performed using commercial statistical software packages.

By performing a spatial analysis of the data, the details of the risk assessment

might be further refined. For example, one component of a fish advisory is often

the establishment of risk-based consumption limits (see Volume 2 of this series).

In order to calculate these limits, an estimate of the contaminant concentration in

the target species must be available. In the example shown in Table N-1, there
APPENDIX N

N-7

are three estimates of cadmium concentration. A spatial analysis of these data

can help to identify which of the concentrations (if any) to use in establishing riskbased consumption limits.

The initial steps in the flowchart on Figure N-1 are to determine whether

parametric or nonparametric statistical tests should be used. The first step is to

test whether each of the three groups of data are from populations that are

normally distributed. Three tests that may be used for this purpose are the

Kolmogorov-Smirnov test for normality (Massey, 1951), Shapiro and Wilk’s W test

(Shapiro et al., 1968; Royston, 1982), and Lilliefors’ test (Lilliefors, 1967). The

results for the W test on each of the three groups of data indicate that each group

was sampled from populations that are normally distributed (Table N-1). The next

step is to test for homogeneity of variances between the three groups. Three

tests that may be used for this purpose are Levene’s test (Milliken and Johnson,

1984), the Hartley F-max test (Sokal and Rohlf, 1981), and the Cochran C test

(Winer, 1962). The result of Levene’s test indicates that the variances of the three

groups of data are not significantly different from each other (Table N-1). These

test results mean that parametric statistics (the left side of Figure N-1) are

appropriate for this dataset. 

An appropriate parametric test to perform to determine whether the three mean

cadmium concentrations are significantly different from each other is a 1-way

ANOVA. The result of this test indicates that the three means are significantly

different (Table N-1). What this result does not show, however, is whether each

mean concentration is significantly different from both of the other mean

concentrations. For this answer, multiple comparison tests can be used to

perform all possible pairwise comparisons between each mean. 

Three tests that can be used to perform a multiple comparison are the NewmanKeul test (Sokal and Rohlf, 1981), Duncan’s Multiple Range test (Hays, 1988;

Milliken and Johnson, 1984), and the Tukey Honest Significant Difference test

(Hays, 1988; Milliken and Johnson, 1984). Three pairwise comparisons are

possible between three means (1 vs. 2, 1 vs. 3, and 2 vs. 3). The results of

Duncan’s Multiple Range test indicate that the mean concentration at station 1

(21.5 ppm) is significantly lower than the mean concentrations at both station 2

(29.4 ppm) and station 3 (31.3 ppm), which in turn are not significantly different

from each other. Therefore, to be most conservative (i.e., protective), the state

could use the mean of the 16 replicate samples from stations 2 and 3 to calculate

risk-based consumption limits. In this example, use of the concentration from any

single station would not truly represent the potential contaminant exposure to fish

consumers in the waterbody of concern.

N.2 TEMPORAL COMPARISON OF STATIONS

Both screening and intensive studies are often repeated over time to ensure that

public health is adequately protected. By examining monitoring data from several

time periods from a single site, it may be possible to detect trends in contaminant
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concentrations in fish tissues. Trend analysis data should never be used to

conduct risk assessments. Procedures for conducting risk assessments are

adequately covered elsewhere in this document (see Section 6.1.2.7). Trend

analysis may, however, be useful for monitoring the effects of various environmental changes or policies on the contaminant concentrations in the target

species. For example, a state may have issued a fish advisory for a contaminant

for which the source is known or suspected. Source control for this contaminant

is the obvious solution to the environmental problem. An evaluation of the

effectiveness of the source control may be made easier by trend analysis. The

state would still need to perform statistical calculations comparing data from each

sampling site to the selected SV, but trend analysis could yield valuable

information about the success of remediation efforts even if the fish advisory

remained in place because of SV exceedances.

Trend analysis can be performed using the statistical framework outlined in

Figure N-1, but complexities in pollution data collected over time may make this

approach unsuitable in some instances. The types of complexities for which other

statistical approaches might be warranted can be divided into four groups: (1)

changes in sampling and/or analysis procedures, (2) seasonality, and (3)

correlated data (Gilbert, 1987). Each of these subjects is discussed briefly here.

Changes in the designation of an analytical laboratory to perform analyses or

changes in sampling and/or analytical procedures are not uncommon in long-term

monitoring programs. These changes may result in shifts in the mean or variance

of the measured values, which could be incorrectly attributed to natural or

manmade changes in the processes generating the pollution (Gilbert, 1987).

Ideally, when changes occur in the methods used by the monitoring program,

comparative studies should be performed to estimate the magnitude of these

changes.

Seasonality may introduce variability that masks any underlying long-term trend.

Statistically, this problem can be alleviated by removing the cycle before applying

tests or by using tests unaffected by cycles (Gilbert, 1987). Such tests will not be

discussed here. States interested in performing temporal analyses with data for

which a seasonal effect is hypothesized should consult the nonparametric test

developed by Sen (1968) or the seasonal Kendall test (Hirsch et al., 1982).

Measurements of contaminant concentrations taken over relatively short periods

of time are likely to be positively correlated. Most statistical tests, however,

including those in Figure N-1, require uncorrelated data. Gilbert (1987) discusses

several methods for performing the required analyses in these cases.

Temporal trends in contaminant concentrations may be detected by regression

analyses, whereby the hypothesis is tested that concentrations are not changing

in a predictable fashion (usually linear) over time. If the hypothesis is rejected, a

trend may be inferred. States interested in performing regression analyses should
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consult statistics textbooks such as Gilbert (1987) or Snedecor and Cochran

(1980).

N.3 REFERENCES

Gilbert, R.O. 1987. Statistical Methods for Environmental Pollution Monitoring.

Van Nostrand Reinhold Company, New York, NY. 320 pp.

Hays, W.L. 1988. Statistics. Fourth Edition. CBS College Publishing, New York,

NY.

Hebert, C.E. and K.A. Keenleyside. 1995. To normalize or not to normalize? Fat

is the question. Environmental Toxicology and Chemistry 14(5):801-807.

Hirsch, R.M., J.R. Slack, and R.A. Smith. 1982. Techniques of trend analysis for

monthly water quality data. Water Resources Research 18:107-121.

Lilliefors, H.W. 1967. The Kolmogorov-Smirnov test for normality with mean and

variance unknown. J. Amer. Stat. Assoc. 62:399-402.

Massey, F.J., Jr. 1951. The Kolmogorov-Smirnov test for goodness of fit. J.

Amer. Stat. Assoc. 46:68-78.

Milliken, G.A., and D.E. Johnson. 1984. Analysis of Messy Data: Volume 1.

Designed Experiments. Van Nostrand Reinhold Company, New York, NY.

Royston, J.P. 1982. An extension of Shapiro and Wilk’s W test for normality to

large samples. Applied Statistics 31:115-124.

Sen, P.K. 1968. On a class of aligned rank order tests in two-way layouts.

Annals of Mathematical Statistics 39:1115-1124.

Shapiro, S.S., M.B. Wilk, and H.J. Chen. 1968. A comparative study of various

tests of normality. J. Amer. Stat. Assoc. 63:1343-1372.

Snedecor, G.W., and W.G. Cochran. 1980. Statistical Methods. 7th edition.

Iowa State University Press, Ames, IA.

Sokal, R.R., and F.J. Rohlf. 1981. Biometry. The Principles and Practice of

Statistics in Biological Research. Second Edition. W.H. Freeman and

Company, New York, NY. 859 pp.

Winer, B.J. 1962. Statistical Principles in Experimental Design. McGraw-Hill,

New York, NY.